HK40045065A - Image analysis and measurement of biological samples - Google Patents

Description

Image analysis and measurement of biological samples

Background information

Analysis of a biological sample from a subject can be important for diagnosis, monitoring, and/or treatment of a subject with respect to the health of the subject. There are a number of known methods available for the analysis of biological samples. However, there is also a need for improvements in the analysis of biological samples in order to provide better diagnosis, monitoring, and/or treatment of subjects.

Lead clause

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

The methods, devices, systems, and apparatus described herein are used for optical and image analysis and/or measurement of biological samples.

Examples of the invention herein include sample holders adapted to hold samples, including biological samples, for optical inspection, optical measurement, and other inspection and measurement. In an example, a sample holder is provided having an optically transmissive portion and a portion designed to provide internal light reflection within the sample holder. In an example, the internal reflection may include partial internal reflection and may also include complete internal light reflection. Incident light from an external light source and incident from the side of the sample holder can effectively illuminate a sample within the sample holder from multiple directions. In examples, an external light source placed on one side of the sample holder may provide epi-illumination for a sample within the sample holder; possibly providing transmissive illumination for a sample in the sample holder; or may provide both epi-illumination and transmission illumination for a sample in the sample holder.

Examples of the invention herein include systems in which sample holders adapted to hold samples are contained. Such systems are suitable for inspecting and measuring samples, including biological samples, by methods such as optical inspection, optical measurement, but also for other inspections and measurements. In the example, a system is shown herein that includes a sample holder having an optically transmissive portion and a portion designed to provide internal light reflection within the sample holder. In an example, the internal reflections within the sample holder of one of the systems shown herein may include partial internal reflections and may also include total internal reflection of light rays. The system shown here may include a light source. A light source from outside a sample holder and incident light entering from the side of the sample holder can efficiently illuminate a sample in the sample holder from multiple directions. In an example, a light source disposed on an exterior side of the sample holder may provide epi-illumination for a sample within the sample holder; possibly providing transmissive illumination for a sample in the sample holder; and/or may provide both epi-illumination and transmission illumination for a sample in the sample holder. The system shown here may include one detector or several detectors; these detectors may include optical detectors and may also include other detectors. The detectors are adapted and designed to measure a sample in a sample holder, the subject and characteristics of a sample, and the object in a sample; these measurements may include qualitative and quantitative measurements. Examples of systems shown herein may include filters, apertures, gratings, lenses, and other optical elements. Examples of systems shown herein may include mechanical means for positioning, moving, and adjusting a sample holder, a light source, a lens, a filter, or other elements or components of a system shown herein. The example systems shown herein may include components or elements for transporting, dispensing, holding, heating, mixing, staining, conditioning, or preparing, processing, or modifying a sample. The example system shown herein may include components and elements for transporting, protecting, filling, or otherwise manipulating a sample holder. The example systems shown herein may include components and elements to physically manipulate and process a sample, as well as to physically manipulate a sample rack; and these components and elements may include, but are not limited to, a pipette, a pump, a centrifuge, other container mechanisms for moving and manipulating a sample, a sample holder, pipette tips, and reagents for use with a sample or other parts. The example systems shown herein may include components and elements for chemical analysis, including nucleic acid analysis, protein analysis, general chemical analysis, electrochemical analysis, and other analysis of a sample or portion of a sample.

The sample holders and systems shown herein may be used anywhere; the methods shown herein may be practiced anywhere including a clinical laboratory, a research laboratory, a clinic, a hospital, a doctor's office, a point-of-care service station, and any other suitable location. The samples held by the sample holders shown herein, and the samples tested using the systems and methods shown herein, include any biological sample, and may be small volumes of biological samples. In an example, a sample may be a small volume of blood or urine sample, and the volume may be less than about 250 μ L, or less than about 150 μ L, or less than about 100 μ L, or less than about 50 μ L, or less than about 25 μ L, or less than about 15 μ L, or may be equal to or less than the volume of blood obtained from a single fingertip blood draw.

In one example, a method is provided for measuring a component of interest in a cell population in a sample, comprising: a) quantitatively measuring a marker expressed in cells of the cell population in the sample; b) determining, with the aid of a computer, the approximate number of cells contained in the population of cells in the sample based on the measurements of part a); c) selecting a dose of an agent to be added to the sample based on the results of part b), the agent specifically binding to the component of interest in the cells of the population and being detectable at any time; d) adding a selected dose of reagent to the sample based on the results of part c); e) detecting cells within the sample for detection of binding of the agent to the component of interest; and f) determining the amount of the component of interest in the cells of the cell population in the sample based on the amount of the agent that binds to the component of interest. In one example of the method, the agent in part c) is an antibody.

The present application further herein shows a method of measuring a component of interest in cells of a cell population in a sample, comprising: a) (ii) performing a quantitative measurement of a marker, or a cellular characteristic, expressed in cells of the population of cells in the sample; b) determining, with the aid of a computer, the approximate number of cells contained in the population of cells in the sample based on the measurements of part a); c) adding to the sample an amount of a cellular marker, said amount of cellular marker being dependent on the results of part b), and said cellular marker specifically binding to a component of interest in cells of the population of cells and being detectable at any time; d) detecting cells in the sample for binding of the marker to the component of interest; and e) determining the amount of the component of interest in the cells of the cell population in the sample based on the amount of the label bound to the component of interest.

In another example, a method for focusing a microscope is provided, comprising: a) mixing a sample containing a microscopic analysis target with a reference particle of known size to produce a mixture containing the sample and the reference particle; b) exposing the mixture obtained in step a) in the path of a microscope light; c) exposing the mixture obtained in step a) to a light beam for observing the reference particle; and d) focusing the microscope according to the position of the reference particle within the mixture, or according to the sharpness of the image of the reference particle.

In yet another example, provided herein is a method of distinguishing one of a plurality of cells from a sample containing the cells, comprising: a) detecting one of the plurality of cells for at least one of: (i) expression of a cell surface antigen; (ii) the amount of one cell surface antigen; (iii) cell size; b) detecting the cells of a) for at least one of the following purposes: (i) the size of the cell nucleus; (ii) nuclear morphology; c) detecting the cells in a) and b) for quantitative cellular light scattering, wherein the combined information from a), b) and c) is used to distinguish between cells in the sample containing a plurality of cells.

In yet another example, provided herein is a system comprising a detector assembly for use with a sample holder for holding a sample to be tested. In one non-limiting example, the sample holder is a cuvette, with structures and/or materials therein that enable the cuvette to be engaged and moved from a position to the detector assembly. In some examples, the probe assembly has a first surface for engaging a surface of the sample holder in such a manner that the interface between the probe assembly and the sample in the sample holder does not optically interfere with the optical path from the probe assembly to the sample in the sample holder. In one example, the detector assembly may have more than one position corresponding to one or more sample holders. Some instances may have the same sample holder for each position. Alternatively, some instances may have different sample holders corresponding to at least some of the positions associated with the detector assembly.

In one example shown herein, a sample holder, such as but not limited to a cuvette having optical, dimensional, material, and/or physical properties, is provided to allow it to receive the sample for analysis through the detector assembly while maintaining the sample physically separate and not in direct contact with the detector assembly. This can be very useful for liquid samples containing a tangible component.

In one example shown herein, the detector assembly may be a multichannel microscope unit designed to detect, acquire, or measure the shape, physical, optical, and biochemical characteristics of a cell or cells in a sample, all within the same device. It can provide both quantitative and descriptive information. One example of the detector assembly may use multiple markers of the same color and wavelength, and the detector assembly is designed to de-spin the signals from the markers within the sample (e.g., binding to cells within the sample), thereby reducing the number of spectral traces and light sources required within the assembly.

It should be understood that some examples herein may include a sample holder, such as, but not limited to, a cuvette, of a material having physical characteristics that increase dark field illumination, and some of which may be configured to provide light reflectance (including, but not limited to, light reflectance within the cuvette), and some of which may be selectively configured for mechanical support; some structures in the examples may provide both mechanical support and light reflection. In examples, a sample holder is designed to provide transmissive illumination for a sample by light reflection within the sample holder. In examples, a sample holder is designed to provide transmissive illumination for a sample by light reflection within the sample holder; this reflection may include Partial Internal Reflection (PIR), and this reflection may include Total Internal Reflection (TIR). In an example, a sample holder is designed to provide transmitted illumination for a sample by light reflection within the sample holder, where the reflected light source is located on the same side of the sample holder as the optical system for detecting and measuring light (e.g., the light source is an epi-illumination light source).

The system herein can use both epi (direct) and transmission (reflected) illumination in dark field imaging. This is in contrast to conventional dark field imaging, which uses epi-illumination or perspective illumination, rather than both types of illumination, and does not provide both types of illumination from a single light source or single direction or location. Thus, the combination of epi-illumination and transillumination shown here differs from known systems in that both the transillumination and the epi-illumination are generated from the same light source. Alternatively, a shaped sample holder, such as a cuvette, may be used to provide the transmitted illumination. In an example, one sample holder is designed to provide transmissive illumination by light reflection. In an example, one sample holder is designed to provide transmitted illumination by light reflection within the sample holder. In examples, one or more dimensions, shapes, surfaces, materials, or other structures of a shaped sample holder can be effective to provide internally reflected light within the shaped sample holder. In examples, one or more dimensions, shapes, surfaces, materials, or other structures of a shaped sample holder may be effective to provide Partial Internal Reflection (PIR) light within the shaped sample holder. In examples, one or more dimensions, shapes, surfaces, materials, or other structures of a shaped sample holder can be effective to provide Total Internal Reflection (TIR) light within the shaped sample holder. Alternatively, the intensity of the transmitted illumination is not negligible. In an example, a shaped sample holder may include a reflective surface effective to increase the intensity of transmitted illumination. The dark field light source may be a Light Emitting Diode (LED), laser, or other illumination source that provides the desired illumination and/or excitation wavelength.

In one example, there is a physical distance between the combination of the microscope objective and the light source, such as but not limited to an annular light (for dark field microscopy), forming a compact space for the detector assembly. In one example, only light at a desired wavelength or within a desired wavelength range can be directed to the sample. In one example, the light is unpolarized light. In another example, the light is polarized light.

In yet another example, information obtained from flow cytometry, whether from the sample preparation stage or the analysis stage, is used to indicate and/or trigger a subsequent procedure. In an example, such a secondary operation may be to remind us to perform a manual review directly. In examples, such a secondary procedure may be to use an estimated cell count or other information obtained in the sample preparation step to guide the performance of a test, which may be a later step in the procedure, or may be a test of other procedures.

Techniques for counting cells may also provide methods for processing sample holders with asymmetric shapes and/or chamber surfaces. One method comprises the use of: a) a volumetric metering tube technique introduces a known volume of a sample into an analysis area, such as a tube in a sample holder. The method may include counting cells within the sample holder. Since we know the volume of the sample, we will also know the concentration of cells in the volume (this operation may be performed in a sample holder with a hydrophobic container or cuvette or chamber with a similar surface). Another method comprises the following steps: b) a ratio-based metrology technique mixes the sample with a known number of beads and calculates the cell concentration in the sample based on the observed number of beads.

In yet another example described herein, a method is provided that includes measuring a physical blood component, such as, but not limited to, measuring Red Blood Cell (RBC) volume in a blood sample, assuming the RBC to be approximately spherical and measuring the RBC volume using a dark field microscope.

In yet another example described herein, a method is provided that includes measuring platelet volume. The method may include labeling platelets with a fluorescent dye and measuring the observed platelet size; adding a ball of known size to the sample; the observed bead image size is compared to the platelet image, the bead is used as a calibration to determine the size of the platelets, and the volume of platelets in the sample is determined.

In still further examples described herein, methods are provided for cytomorphological detection and measurement of a sample; measurement of cell number; detecting particles; measurement of the number of particles; detecting crystals; measurement of the number of crystals; detection of cell aggregation; measurement of the number of cell aggregates, and other characteristics and quantitative analysis in a sample.

In view of this, the present patent application shows:

a system for analyzing a sample, the system comprising: a sample holder having a sample chamber for receiving the sample; at least one portion of the sample holder contains an optically transmissive material; the optically transmissive material has an optically transmissive surface and a reflective surface; an illumination source for providing light for illuminating and penetrating said optically transmissive surface; (ii) light from said illumination source simultaneously provides both epi-illumination and transmission illumination of a sample in the sample holder, wherein light constituting the epi-illumination is directed from said illumination source to said sample without being reflected at a surface of the optically transmissive material of the sample holder; the light rays constituting the transmitted illumination travel in the optically transmissive material and reach the sample after at least one reflection by at least one surface of said optically transmissive material. In an example, a sample holder of a system containing the structure shown herein may have an elongated tube for holding a cuvette for a sample. In an example, the sample holder may have one or more non-optically transmissive surfaces.

In the example of systems shown herein, the transmitted illumination is provided at least in part by internally reflected light from a surface, and may be provided at least in part by totally internally reflected light within the cuvette. In the example of the system shown here, the transmitted illumination is provided at least in part by internally reflected light from a surface, and possibly at least in part by internally reflected light within the cuvette.

In one example, a sample rack may contain two or more sample chambers for holding samples. A sample holder, such as a cuvette containing the structure shown herein, may have a rectangular horizontal cross-sectional shape; may have a circular horizontal cross-sectional shape; may have a saw-tooth like vertical cross-sectional shape; may have a stair-like vertical cross-sectional shape; or may have other shapes.

In examples, a sample holder may be movable relative to an illumination source and may be movable to a plurality of positions, wherein an optically transmissive surface of the sample holder may be illuminated by the illumination source at each position.

In an example, an illumination source may include an annular light. In an example, a ring light may be selected from a Light Emitting Diode (LED) based ring light and a laser based ring light.

In an example, a system as shown herein may include a support structure having an optically transmissive surface in shaped engagement with the optically transmissive surface of the sample holder.

In the example, a system as shown herein may include a pressurizing device designed to hold the sample holder in a desired position for illumination by the illumination source.

In an example, a system as shown herein may include a detector designed to image at least a portion of a tube within the sample holder.

In the example, a sample holder as shown herein may include an elongated conduit designed to receive at least a portion of a sample.

In examples, a sample holder as shown herein may be designed to hold the sample in a stationary, non-flowing state during imaging; in an example, a sample holder may be designed to hold a portion of a sample in a stationary, non-flowing state while another portion is in a flowing state.

In examples, an illumination source as shown herein may be movable relative to the sample holder.

In examples, a sample holder as shown herein may be designed to hold the sample in a fluid state during imaging.

In an example, a sample holder as shown herein may include a liquid circuit completely confined within the sample holder, wherein the sample is located within said liquid circuit, effectively keeping the sample separate from said probe.

In examples, a sample holder as shown herein may be movable relative to the detector. In an example, a detector as shown herein may be movable relative to the sample holder.

In an example, a sample holder and an illumination source as shown herein contain at least part of the optical analysis unit, and the system further comprises a clinical analysis unit to perform clinical analysis on a sample.

In an example, a system as shown herein is designed to dispense a sample into an optical analysis unit and a clinical analysis unit, effectively enabling the clinical analysis unit and the optical analysis unit to perform optical analysis and clinical analysis of a portion of a sample at the same time. In an example, such a clinical assay may be selected from common chemical assays, nucleic acid assays and enzyme-linked assays.

In an example, a system as shown herein may comprise a plurality of clinical analysis units, wherein each such clinical analysis unit is adapted to provide a clinical analysis selected from the group consisting of general chemical analysis, nucleic acid analysis and enzyme linked analysis.

The present application further provides a cuvette comprising a sample chamber for receiving a sample, said cuvette comprising at least one portion comprising an optically transmissive material; the optically transmissive material has an optically transmissive surface and a reflective surface; wherein said optically transmissive surface and said optically reflective surface are operatively configured such that light transmitted through the optically transmissive surface simultaneously provides both epi-illumination and transmission illumination of said sample in the sample chamber, wherein light constituting the epi-illumination is directed from said illumination source to said sample without being reflected from a surface of the optically transmissive material of the sample holder; the light rays constituting the transmitted illumination travel in the optically transmissive material and reach the sample after at least one reflection by at least one surface of said optically transmissive material.

In the example, a cuvette is shown here with a sample chamber containing an elongated channel. In the example, one cuvette shown here contains two or more sample chambers for receiving samples.

In an example, one cuvette shown here may have one or more non-optically transmissive surfaces.

In the examples shown herein, the transmitted illumination that may be provided in a cuvette is, at least in part, internally reflected light within the cuvette. In the examples shown herein, transmitted illumination, which may be provided within a cuvette, is at least partially reflected light within a portion of a surface of the cuvette. In the examples shown herein, the transmitted illumination that may be provided within a cuvette is, at least in part, totally internally reflected light from a surface of the cuvette.

In the example, a cuvette as shown here may have a rectangular horizontal cross-sectional shape; in the example, a cuvette as shown here may have a circular horizontal cross-sectional shape. In the examples, a cuvette as shown herein may have a sawtooth-like vertical cross-sectional shape; in the example, a cuvette as shown here may have a staircase-like vertical cross-sectional shape.

The invention application shows a method: for example, the present application shows a method for distinguishing a cell from a sample containing a plurality of cells: (a) placing said sample in a sample holder in a sample chamber for receiving the sample, said sample holder having at least one portion comprising an optically transmissive material; the optically transmissive material has an optically transmissive surface and a reflective surface; wherein said optically transmissive surface and said optically reflective surface are operatively configured such that light transmitted through the optically transmissive surface simultaneously provides both epi-illumination and transmission illumination of said sample in the sample chamber, wherein light constituting the epi-illumination is directed from said illumination source to said sample without being reflected from a surface of the optically transmissive material of the sample holder; the light rays forming the transmission illumination run in the optical transmission material and reach the sample after being reflected for at least one time by at least one surface of the optical transmission material; (b) the sample holder illumination may be effective to provide both epi-illumination and transmission illumination of the sample; (c) a cell within the sample is identified. In the examples, several methods are shown herein, including the identification method, involving the identification of the cells by a probe designed to image at least a portion of the sample chamber. In the example shown here, a sample chamber used in this method may contain an elongated tube.

The present application further shows a method of focusing a microscope, comprising: a) mixing a sample containing a microscopic analysis target with a reference particle of known size effective to produce a mixture containing the sample and the reference particle; b) placing the mixture obtained in step a) in the path of a microscope illumination; c) placing the mixture obtained in step a) under a light beam for observing the reference particle; d) focusing the microscope according to the position of the reference particle within the mixture, or according to the sharpness of the image of the reference particle.

The present application shows a method of identifying a cell from a sample containing a plurality of cells, comprising: (a) detecting a cell of the plurality of cells for at least one of: (i) expression of a cell surface antigen; (ii) the amount of one cell surface antigen; (iii) cell size; b) detecting the cells in a) for at least one of the following purposes: (i) the size of the cell nucleus; (ii) nuclear morphology; c) detecting the cells in a) and b) for quantifying cellular light scattering, and identifying the cells in the plurality of cell samples using the combined information from a), b), and c).

In at least one example described herein, a system for imaging a sample, the system comprising: a sample container containing said sample, a platform containing a sample container receptacle having an optically transmissive surface; a light source for illuminating through the platform the tangible elements within the sample, wherein the sample container has an interface surface for engaging the optically transmissive surface of the sample container receiver, and wherein the interface surface conforms to the optically transmissive surface without significant distortion when light passes through the interface surface.

It is to be understood that the examples herein may be designed to include one or more of the following structures. For example, the interface surface of the sample container may be formed from a high molecular polymer material. Alternatively, this may be a light transmissive material. Alternatively, the material forming the sample vessel interface surface may use a material that is softer than the optically transmissive surface of the sample vessel receiver. Optionally, a pressing unit is provided for pressing the interface surface to conform to the shape of the optically transmissive surface of the sample container receiver. Alternatively, a processing unit may be designed to couple with the sample container, assist in transporting the sample container up and down the platform, and increase the mechanical strength of the sample container. Alternatively, the processing unit may be a light-tight unit designed to couple with the sample container. Alternatively, the processing unit may be formed with physical structures, protrusions or the like to facilitate engagement with a robotic manipulator, pipette unit, or other mechanical carrier. Alternatively, the processing unit may be formed of magnetic, electromagnetic, or other properties that facilitate engagement and/or disengagement. Alternatively, all images of the sample may be completed without the necessity of passing substantially straight light through one surface and out the opposite surface to a detector. Alternatively, the light source need not be located on one side of the sample container but transmits light to a detector on the opposite side of the sample container.

It is to be understood that embodiments of the invention may be adapted to one or more of the structures described in this invention.

This abstract is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This abstract is not intended to identify key or essential structures of the claimed subject matter, nor is it intended to be used to limit the claimed subject matter.

Brief description of the drawings

FIG. 1 shows: (A) plotting the side scatter intensity (X-axis) against the fluorescence intensity of a mixed cell containing fluorescently labeled natural killer cells and neutrophils, the fluorescent label recognizing CD 16; (B) bar graphs show the ratio of nuclear area to all cellular areas of Natural Killer (NK) and neutrophils (Neu); (C) natural killer cells stained with anti-CD 16 antibody (left bar) and cell nucleus (right bar); (D) neutrophils stained with anti-CD 16 antibody (left bar) and anti-nuclear staining (right bar).

FIG. 2 shows: (A) platelets labeled with fluorescent conjugated anti-CD 41 and CD61 antibodies (bright spots); (B) intensity distribution images of fluorescently labeled platelets under 10-fold (left) and 20-fold (right) magnifications; (C) the image of the fluorescence labeled platelet intensity distribution shows the measured intensity (light gray) and a curve fitted to the measured intensity (dark gray).

FIG. 3 shows: the graph shows the relationship between the nominal diameter m (x-axis) of the standard particle and the measurement a.u (Y-axis) based on the fluorescence intensity. (y-axis). The figure also shows the number of balls represented at different points along the curve.

FIG. 4 shows: spherical red blood cells in the cuvette imaged by dark field microscopy, (A) the cuvette only receives epi-illumination; (B) the cuvette received mixed illumination of both epi-and transmission.

FIG. 5 shows: (A) anti-CD 16 antibody staining and nuclear staining of rod-shaped neutrophils (B) anti-CD 16 antibody staining and nuclear staining of lobulated nuclear neutrophils.

FIG. 6A shows an example of an optical system suitable as part of the apparatus or system shown herein and suitable for use in the method shown herein, comprising a typical optical system (e.g., a light source shown as an annular light and an objective lens), cuvettes, and a support structure for receiving and positioning a cuvette for imaging. In this example, the colorimeter v has a rectangular horizontal cross section.

FIG. 6B shows an example of an optical system suitable for use as part of the apparatus or system shown herein and suitable for use in the methods shown herein, including exemplary optical systems (e.g., a light source shown as an annular light and an objective lens), cuvettes, and a support structure for receiving and positioning a cuvette for imaging. In this example, the cuvette has a circular horizontal cross-section.

FIG. 7A shows an example of elements of an optical system suitable for use as part of an apparatus or system as shown herein and suitable for use in the method as shown herein.

FIG. 7B shows an example of optical system components suitable for use as part of the apparatus or system shown herein and suitable for use in the method shown herein, including a telephoto lens and an aperture suitable for limiting the angular range of scattered light reaching a detector.

FIG. 8A provides an example view of an optical system including a support structure holding a cuvette to image a sample, where light from an annular light illumination system falls directly on the sample (epi-illumination); but the light may also be reflected through some structure of the cuvette, thereby providing transmitted illumination. In this example, the cuvette has a stepped vertical cross-section.

FIG. 8B provides an example view of an optical system including a support structure holding a cuvette to image a sample, where light from an annular light illumination system falls directly on the sample (epi-illumination); but the light may also be reflected through some structure of the cuvette, thereby providing transmitted illumination. As shown, an incident ray may be totally reflected by one surface (total internal reflection, TIR), or a portion of an incident ray may be reflected by one surface (partial internal reflection, PIR). In this example, the cuvette has a vertical cross-section that is serrated.

FIG. 8C shows an example of an optical system suitable for use as part of the apparatus or system shown herein and suitable for use in the methods shown herein, including exemplary optical systems (e.g., a light source shown as an annular light and an objective lens), cuvettes, and a support structure for receiving and positioning a cuvette for imaging. In this example, the cuvette includes structures that affect the path of light that strikes the cuvette and the sample within the cuvette.

FIG. 8D shows an example of an optical system suitable for use as part of the apparatus or system shown herein and suitable for use in the methods shown herein, including exemplary optical systems (e.g., a laterally illuminated light source), cuvettes, and a support structure for receiving and positioning a cuvette for imaging. In this example, the cuvette includes structures that affect the path of light that strikes the cuvette and the sample within the cuvette.

FIG. 8E provides a flow chart showing the transfer of a cuvette from a sample preparation site to a sample viewing position proximate an optical detector (labeled D).

FIG. 8F provides a further, detailed flow diagram showing a system including a transport mechanism for transporting a cuvette from a sample preparation site to a sample viewing position proximate an optical detector.

FIG. 9 is a blended image showing blood cell images obtained from whole blood using different imaging techniques and staining. FIG. 9A is a dark field image; FIG. 9B is a photograph showing fluorescently labeled anti-CD 14 antibody attached to monocytes; FIG. 9C is an image showing fluorescently labeled anti-CD 123 antibody attached to basophils; FIG. 9D is an image showing fluorescently labeled anti-CD 16 antibody attached to neutrophils; FIG. 9E is an image showing fluorescently labeled anti-CD 45 antibody attached to leukocytes; FIG. 9F is a view showing cell nucleiStained white blood cell and platelet cell images (red blood cells lack nuclei and cannot be stained with red blood cells)Dyeing).

FIG. 10 is a mixed image showing a blood cell image obtained from whole blood, showing a monocyte, a lymphocyte, an eosinophil, a basophil, and a neutrophil.

FIG. 11 shows a graph of fluorescence detected from cells labeled with different markers (labeled antibodies directed against different cell surface markers or other markers); this multiple labeling approach is useful for identifying cells. FIG. 11A shows identification of monocytes by plotting labeled CD14 intensity (FL-17) and scatter intensity. FIG. 11B shows the identification of basophils by plotting labeled CD123 intensity (FL-19) and CD16 intensity (FL-15). FIG. 11C shows lymphocyte identification by plotting labeled CD16 intensity (FL-15) and CD45 intensity (FL-11). FIG. 11D shows identification of neutrophils by plotting labeled CD16 intensity (FL-15) and scatter intensity (FL-9).

FIG. 12 shows a comparison of the cell count (measured on a aliquot of the same blood sample) obtained using the current method and using other methods (using a commercial whole blood analyzer). Fig. 12A graphically shows the white blood cell count obtained from the current method and the white blood cell count obtained using a commercial complete blood counter. FIG. 12B is a plot showing the red blood cell count obtained from the current method and the red blood cell count obtained using a commercial complete blood count machine. Fig. 12C is a plot showing the platelet count obtained from the current method and the platelet count obtained using a commercial complete blood counter. Figure 12D is a plot showing the neutrophil count obtained from the current method and the neutrophil count obtained using a commercial complete blood counter. Figure 12E plots the monocyte counts obtained from the current method and the monocyte counts obtained using a commercial complete blood counter. Fig. 12F is a plot showing lymphocyte counts obtained from the current method and lymphocyte counts obtained using a commercial complete blood counter.

Detailed Description

It may be helpful to understand that all of the intended scope and advantages of the present apparatus, systems and methods may be described and illustrated in the following patents, such as U.S. patent 7,888,125; U.S. Pat. nos. 8,088,593; us patent 8,158,430; us patent 8,380,541; U.S. patent application serial No. 13/769,798, filing date 2013, 2 month 18; U.S. patent application serial No. 61/802,194, filing date 2013, 3 months and 15 days; U.S. patent application serial No. 13/769,779, filing date 2013, 2 month 18; U.S. patent application serial No. 13/244,947, filing date 2011, 9 months and 26 days; PCT/US 2012/57155, filing date 2012, 9 months 25; U.S. application serial No. 13/244,946, filing date 2011, 9/26; U.S. patent application 13/244,949, filing date 2011, 9/26; and U.S. application serial No. 61/673,245, filed 2011 on 26/9, all of which are incorporated herein by reference.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed. It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, a concept such as "a material" may include a mixture of several materials; the concept of "a component" may include a plurality of components, and so on. The references cited herein are fully incorporated herein unless their coverage is at least partially inconsistent with the express specification.

In the specification and claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

"optional" or "optionally" means that the subsequently described circumstance may or may not occur, and so its description includes instances where the circumstance occurs and instances where it does not. For example, if an apparatus "optionally" contains a structure for a sample collection unit, this means that the sample collection unit may or may not be present, and thus the description includes both apparatus structures having the sample collection unit and apparatus structures not having the sample collection unit.

As used herein, the term "substantial, large" means greater than a minimum or insignificant amount; and "appreciably" means more than a minimum or slightly. Thus, for example, the phrase "substantially different" as used herein indicates that there is a significant height difference between two values, and thus one skilled in the art would consider the difference between the two values to be statistically significant over the range of features measured by the values. Thus, the difference between these two values that differ greatly from each other is typically greater than about 10%, and may be greater than about 20% as a function of a reference or comparison value; greater than about 30%, greater than about 40%, or greater than about 50%.

As used herein, "internal reflection" refers to reflection of light within a material (a first material) at a boundary between the first material and another material (a second material). For example, the first material may be a solid, such as a glass or plastic, and the second material may be air. The internally reflected light travels within the first material before being reflected. The internal reflection may be partial (partial internal reflection: PIR) or total (total internal reflection: TIR). Thus, the internal reflection of all incident light rays at one surface back into the first material is TIR; the internal reflection within a first material where not all incident light rays are reflected back at a surface is a PIR. (for PIRs, some light may pass through the boundary and some light is reflected back into the material at the surface.) the angle of incidence is an important factor in determining the degree of internal reflection; it measures the angle between an incident ray and a line perpendicular to the interface. Whether TIR occurs depends on the angle of incident light relative to the interface surface of the first material and the second material, the refractive index of the first material, the refractive index of the second material, and other factors (e.g., the wavelength of light may affect TIR, as the refractive index typically varies with wavelength). The angle at which a ray is totally internally reflected is called the critical angle; incident light rays with an angle of incidence greater than the critical angle will be totally internally reflected (retained within the material: TIR). However, for PIR, some of the incident rays having an angle of incidence less than the critical angle are internally reflected (the remaining rays are refracted and pass out of the first material into the second material).

As used herein, a "sample" may be, but is not limited to, a blood sample, or a urine sample, or other biological sample. A sample may be, for example, a blood sample (e.g., a sample from a fingertip puncture, or venipuncture, or an arterial blood sample, and may be whole blood, serum, plasma, or other blood sample), a urine sample, a pathological specimen, a tissue slice, a stool specimen, or other biological specimen; a water sample, a soil sample, a food sample, an air sample; or other samples (e.g., nasal swab or nasopharyngeal wash, saliva, urine, tears, digestive fluid, spinal fluid, mucus, sweat, cerumen, grease, glandular secretion, cerebrospinal fluid, tissue, semen, cervical secretion, vaginal secretion, synovial fluid, pharyngeal swab, breath, hair, nails, skin, pathological specimen, placental fluid, amniotic fluid, umbilical cord blood, lymph fluid, bodily fluid, sputum, mucus, pus, a microbiological sample, fetal stool, breast milk, and/or other secretions).

Thus, as used herein, a "sample" includes a portion of a blood, urine, or other biological sample, which may be of any suitable size or volume, and which is primarily intended to be of a small size or volume. In some examples of such systems, the tests and methods shown herein may be performed using a small volume of blood sample, or no more than a small fraction of a volume of a blood sample, where the small volume contains no more than about 5 mL; or contains no more than about 3 mL; or contains no more than about 2 mL; or contains no more than about 1 mL; or contains no more than about 500 μ L; or contains no more than about 250 μ L; or contains no more than about 100 μ L; or contains no more than about 75 μ L; or contains no more than about 50 μ L; or contains no more than about 35 μ L; or contains no more than about 25 μ L; or contains no more than about 20 μ L; or contains no more than about 15 μ L; or contains no more than about 10 μ L; or contains no more than about 8 μ L; or contains no more than about 6 μ L; or contains no more than about 5 μ L; or contains no more than about 4 μ L; or contains no more than about 3 μ L; or contains no more than about 2 μ L; or contains no more than about 1 μ L; or contains no more than about 0.8 μ L; or contains no more than about 0.5. mu.L; or contains no more than about 0.3 μ L; or contains no more than about 0.2. mu.L; or contains no more than about 0.1. mu.L; or contains no more than about 0.05 μ L; or contains no more than about 0.01. mu.L.

In an example, the volume of sample collected by fingertip penetration may be, for example, about 250 μ L or less, or about 200 μ L or less, or about 150 μ L or less, or about 100 μ L or less, or about 50 μ L or less, or about 25 μ L or less, or about 15 μ L or less, or about 10 μ L or less, or about 5 μ L or less, or about 3 μ L or less, or about 1 μ L or less.

As used herein, the term "point-of-service location" may include a location at which a subject may receive a service (e.g., examination, monitoring, treatment, diagnosis, instruction, sample collection, ID confirmation, medical services, non-medical services, etc.); and may include, but is not limited to, a subject's home, a subject's work site, a medical service provider's (e.g., doctor's) site, a hospital, an emergency room, an operating room, a clinic, a medical service professional's office, a laboratory, a retail store [ e.g., a pharmacy (e.g., retail pharmacy, clinical pharmacy, hospital pharmacy), a pharmacy, a supermarket, a grocery store, etc. ], a transportation vehicle (e.g., a car, boat, truck, bus, airplane, motorcycle, ambulance, mobile unit, law enforcement vehicle, police vehicle, or other vehicle for transporting a subject from one site to another, etc.), a travel medical services unit, mobile unit, school, nursery center, screening site, competition site, medical assisted living residence, government office, etc, An office building, tent, body fluid sample acquisition site (e.g., a blood collection center), entrance at or near a site that a subject may wish to enter, facility site at or near a facility that a subject may wish to use (e.g., a site where a computer is located if the subject wishes to use the computer), site where a sample processing facility receives a sample, or any other point-of-service site described elsewhere herein.

The term "cell" as used in the context of a biological sample includes samples that generally approximate the size of an individual's cells, including but not limited to vesicles (like liposomes), cells, viral particles, and substances associated with small particles such as beads, nanoparticles, or microspheres.

As used herein, the term "bind" refers to a reaction or interaction between two materials that results in tight binding of the two substances, e.g., a reaction between a ligand and a receptor provides an example of binding where the ligand becomes firmly attached to the receptor, binding between an antibody and its target antigen, and binding of a carrier protein to its cargo, e.g., intrinsic factor and vitamin B, are further examples of reactions of a substance with other substances.

As used herein, the term "binding agent" refers generally to any complex or polymer, such as an antibody, that can be firmly or specifically attached to a target. These binding agents include, but are not limited to, antibodies (polyclonal or monoclonal, antibody fragments, immunoadhesins, and other similar antibody variants and replicates), natural binding proteins (e.g., intrinsic factor protein specific for vitamin B12), ligands that bind to its target receptor, substrates that bind to specific enzymes, binding partners such as avidin and biotin, small molecules that bind strongly and specifically to target molecules, and the like. Bacteria, viruses, synthetic scaffolds, and other substances or materials that bind or adhere to a particular target may be used as binding agents. A binding agent may be, or may include, or may be linked to a label such as a dye, or fluorophore, or other detectable moiety.

As used herein, a "marker" is a detectable substance whose expression causes an object to become visible or detectable; or its presence at a location or place indicates the representation of an object at that location or place. A marker may be used to mark a cell, structure, particle, or other target, and may be useful in discovering, determining expression, localization, confirmation, quantification, or measuring a target or property within a sample. Labels may include, but are not limited to, stains, dyes, ligands, antibodies, particles, and other substances that may bind to or be localized at certain specific objects or locations; bacteria, viruses, or cellular fluids that may grow or localize certain specific targets or sites may also be used as markers; detectable attributes or properties of cells or other targets may also be used as markers.

As used herein, the terms "stain" and "staining" may be used interchangeably to refer to elements, complexes, and macromolecules that allow a substance or component in a sample to be more easily detected than if the substance or component had not been stained or stained. For example, a blood sample treated with a DNA dye such as propidium iodide makes the nucleoli of nucleated cells more visible and makes detection or quantification of such cells easier than if they were not stained, and may even be expressed in non-nucleated cells (e.g., red blood cells).

As used herein, a "detector" may be any device, apparatus or system that provides information from a signal, image or other information related to a subject, such as a sample. The detectable signal or information may include, for example, optical, electrical, mechanical, chemical, physical, or other signals. A detector may be, for example, an optical detector, or an electronic detector, or a chemical detector, or an electrochemical detector, or an acoustic detector, or a temperature detector, or a mechanical detector, or other detectors.

As used herein, an "optical detector" detects electromagnetic radiation (e.g., light). An optical detector may detect an image or be used with an image; or the beam intensity may be detected without regard to the image; or both. An optical detector may detect or measure the intensity of the light. Some optical detectors may be sensitive to, or limited to, detecting or measuring a particular wavelength or range of wavelengths. For example, the optical detector may include a photodiode detector, photomultiplier tube, charge-coupled device, laser diode, spectrophotometer, camera, microscope, or other device capable of measuring light intensity (a single wavelength, multiple wavelengths, or wavelengths within a range, or ranges), forming an image, or both.

The term "ploidy" as used herein refers to the amount of DNA within a cell and the amount of DNA within a cell in a sample. Ploidy measurements provide a measure that does not necessarily take into account whether a cell or a population of cells contains a normal amount of DNA or an abnormal amount of DNA; alternatively, the amount of DNA in the proliferating cell population is normal when the DNA is replicated during cell division and proliferation. Ploidy measurements may be performed by imaging techniques after staining nucleated cells in a sample with a DNA-specific dye.

Quantitative analysis microscopy

In some examples, some methods, systems, and apparatus herein are provided for microscopic quantitative analysis techniques. The microscopic quantitative analysis technique may involve one or more of fluorescence quantitative analysis microscopy, dark field quantitative analysis microscopy, bright field quantitative analysis microscopy, and comparative microscopy for quantitative analysis to measure one or more cellular attributes. Either of these methods may provide morphological information about the cells. This information may be quantitatively measured. In one example, as a microscopic quantitative analysis technique, a sample is analyzed by one or more of fluorescence quantitative analysis microscopy, dark field quantitative analysis microscopy, bright field quantitative analysis microscopy, and comparative microscopic technique at the time of quantitative analysis. The microscopic quantitative analysis technique may include the use of imaging analysis techniques and/or statistical learning and classification methods to process images obtained via microscopic techniques.

A variety of different cellular properties may be measured in microscopic quantitative analysis techniques. Cellular attributes that may be measured include, but are not limited to:

physical properties: for example: cell size, volume, conductivity, low or high angle scattering, and density. Other physical properties that may be measured and/or quantified include, but are not limited to, roundness of a cell or particle, aspect ratio of a cell or particle, perimeter of a cell or particle, convexity of a cell or particle, granularity of a cell or particle, image density of a cell or particle, height of a cell or particle (e.g., size across several focal planes), flatness of a cell or particle, and other physical properties.

Morphological attributes: such as cell shape, area, size and leafiness; nuclear shape, area, size and foliability; mitochondrial shape, area, size and foliability; and the ratio of the cell nucleus volume to the cell volume.

Intracellular properties: such as the distance of the nucleus centroid/cell centroid (i.e., the distance of the nucleus center from the cell center); distance of nuclear lobe centroids of nuclei (i.e., distance of different nuclear lobe centers of nuclei); intracellular protein distribution (i.e., actin, tubulin, etc.); the distribution of intracellular organelles (i.e., lysosomes, mitochondria, etc.); co-localization of proteins with other proteins and organelles, and other properties.

Biochemical properties: such as the level of expression of cellular proteins, cell surface proteins, cytoplasmic proteins, nuclear proteins, cell nucleic acids, cell surface nucleic acids, cytoplasmic nucleic acids, nuclear nucleic acids, cell carbohydrates, cell surface carbohydrates, cytoplasmic carbohydrates, and nuclear carbohydrates.

In some examples, methods, systems, and devices are provided for quantitative measurement of two, three, four, five, or more cellular attributes within a sample, wherein the attributes are selected from the group consisting of physical attributes, morphological attributes, intracellular attributes, and biochemical attributes. In some examples, methods, systems, and devices are provided for quantitative measurement of two, three, four, five, or more cellular attributes within a sample, wherein the attributes are selected from the group consisting of: cell size, cell volume, cell conductivity, cell low angle light scattering, cell high angle light scattering, cell density, cell shape, cell area, cell foliability; nuclear shape, nuclear area, nuclear size, nuclear cladospermia; mitochondrial shape, mitochondrial area, mitochondrial size, mitochondrial leafiness; nuclear volume to cell volume ratio, nuclear centroid/cell centroid distance, nuclear page centroid distance, intracellular protein distribution (i.e., actin, tubulin, etc.); the distribution of intracellular organelles (i.e., lysosomes, mitochondria, etc.); a cellular protein expression level, a cell surface protein expression level, a cytoplasmic protein expression level, a nuclear protein expression level, a cellular nucleic acid expression level, a cell surface nucleic acid expression level, a cytoplasmic nucleic acid expression level, a nuclear nucleic acid expression level, a cellular carbohydrate expression level, a cell surface carbohydrate expression level, a cytoplasmic carbohydrate expression level, and a nuclear carbohydrate expression level.

In some examples, methods are provided for quantitative measurement of two, three, four, five or more cellular attributes within a biological sample by microscopy, wherein the method may include one or more of the following steps or elements: the cellular attributes that are quantitatively measured may be selected from the attributes listed in the immediately preceding paragraph. The biological sample may be pre-treated prior to microscopic analysis. The pre-processing may include any operation that assists the microscope in analyzing the sample, including: processing the sample concentrates cells of interest for microscopic analysis, processing the sample to reduce components in the sample that may interfere with microscopic analysis, adding substances to the sample to assist in microscopic analysis of the sample (e.g., dilution, blocking molecules to reduce non-specific binding of dyes to cells, etc.). Alternatively, a sample may be contacted with one or more binding agents that specifically bind to a cellular component prior to microscopic analysis. The binder may be directly linked to a dye or other particle to reveal the binder. A sample may also be contacted with a second binding agent that binds to the cellular component. A second binder may be attached directly to a dye or other particle to reveal the binder. A sample may be detected in a spectrophotometer prior to microscopic analysis. In conducting a microscopic analysis, a biological sample containing or suspected of containing the microscopic analysis target may be placed in a sample holder, such as a slide or a cuvette. The sample holder containing a sample may be placed in an apparatus intended for microscopic quantitative analysis of the sample. The microscope may be coupled to an image sensor for capturing images generated by the objective lens. In this apparatus, multiple images of a sample may be taken by a microscope. Any one or more of fluorescence quantitative analysis microscopy, dark field quantitative analysis microscopy, bright field quantitative analysis microscopy, and quantitative analysis time contrast microscopy may be used to obtain an image of the sample. Alternatively, the entire sample image in the sample holder may be taken by the microscope. Multiple field of view views of the microscope may be required to take an image of the entire sample within the sample holder. The sample holder may be movable relative to the microscope, or the microscope may be movable relative to the sample holder in order to generate different fields of view to examine different parts of the sample within the sample holder. The sample in the sample rack may be acquired in multiple images within the same field of view. Alternatively, multiple filters may be used in the same type of microscope and the same field of view of the sample to obtain different images of the same sample that contain different information about the sample. Filters that may be used include, but are not limited to, bandpass filters and long-pass filters. A filter may allow certain wavelengths of light to pass through, but block other light from passing through. Alternatively, multiple types of microscopes (e.g., fluorescence, dark field, bright field, etc.) may be used to acquire images of the sample under the same field of view, so that different images of the same sample are acquired that contain different information about the sample. Alternatively, camera technology may be used for microscope image acquisition. Alternatively, the microscope images may be collected in a 3-dimensional format. As with the microscope-implemented methods described herein, the apparatus or system may be configured to link information relating to a cell in one image of the sample with a different image of the same cell in the sample. Multiple attributes of cells in a sample may be detected based on different images of the same sample and/or the same cell. In some aspects, combining attributes/pieces of information of cells within a sample may be used to complete a clinical decision and/or to make certain conclusions about the type of cell that would not be possible if relying only on a single attribute information for the cell.

In some examples, devices and systems are provided for microscopically quantifying two, three, four, five, or more cellular attributes in a biological sample. In some examples, the device or system contains both a microscope or cytometer and a spectrophotometer. The apparatus or system may further comprise a fluid handling device configured to move the sample between a spectrophotometer and a microscope or cytometer. In some examples, apparatus and systems for performing the inventive methods are described in U.S. patent application No. 13/244,947, and U.S. patent application serial No. 13/769,779, which are incorporated herein by reference. Although described above in the context of cells, it is to be understood that some or all of the foregoing may also be applied to crystals, particles, microwires, or other cell-sized objects that may be found in a sample.

Dynamic dilution

In some examples, methods, systems, and apparatus are provided herein for dynamic dilution of a sample containing cells.

As a non-limiting example, a method for dynamic dilution of a sample may include one or more of the following steps or elements to determine a desired amount or concentration of cells or targets within the sample, which information is used as a factor to adjust subsequent sample processing. In this non-limiting example, one or more stains or dyes are added to a biological sample containing cells. The mixture of stain and sample may be incubated. The stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The stained, washed cells may be diluted to a desired volume for further analysis. Such stained, washed cells may be analyzed to determine the approximate number or concentration of cells within the sample or portion of the sample. Depending on the number or concentration of stained cells in the sample or a part of the sample, it may be possible to obtain a volume of sample ready for further analysis, i.e. to obtain the number or concentration of cells required for a further analysis. In some instances, the sample may be diluted as described in U.S. patent application No. 13/355,458, which is fully incorporated by reference herein.

In one example described herein, it is desirable to provide another detection technique, such as, but not limited to, counting cells using fluorescence-based methods and estimating the concentration of cells instead of cell counting. This estimation is described in detail because in order to accurately and repeatedly stain a patient's sample, it is often necessary to selectively titrate the stain (DNA dye/antibody/binding agent, etc.) against a particular number/concentration of cells. For example, a known concentration of stain is applied to a particular number of cells (e.g., 0.2 micrograms of stain per thousand White Blood Cells (WBCs)). After a period of incubation, the sample is washed to remove excess (unbound) dye, set to the appropriate cell density, and imaged.

In this non-limiting example, to estimate the concentration level of a target type of cell, a sample is measured non-destructively using a method different from cell counting, such as but not limited to a spectrophotometer, to allow cell count detection thereof. The method may comprise selecting a marker specific for another cell population of interest. In one non-limiting example, we might select CD20 for B cells. The treatment process comprises labeling the sample with a fluorescent conjugated anti-CD 20 binding agent that is a different color than CD 5. A device, such as but not limited to a fluorescence spectrophotometer, is then used to non-destructively perform a rapid measurement of the fluorescence signal in the sample. Using calibration, it is possible to predict B cell concentrations with limited accuracy to provide an estimated value. In one non-limiting example, such a calibration may relate signal intensity to the number of cells responding to such a signal. Establishing these calibration curves can be used to estimate the number of cells or targets. Other techniques for estimating the number of cells based on an overall signal strength, such as, but not limited to, optical, electronic, acoustic, or the like, are not excluded. From the approximate concentration of B cells, the correct amount and concentration of anti-CD 5 conjugate can be estimated, thus maintaining a proportional relationship between CD5 expression and CD5 fluorescence. In this way, the staining agent and staining process can be optimized/normalized for a particular cell number.

In order to maximize the use of the patient sample obtained (which may be a small volume sample, such as whole blood obtained by taking blood between them, having a volume of 120 μ L or less), it has been necessary to develop methods for counting the number of WBCs contained in a given volume of blood (e.g., to determine the concentration of WBCs/μ L). This allows us to determine, at least estimate, the number of WBCs before adding stain. Once the number is determined, the desired number of cells can be dispensed for incubation with a known concentration of stain to produce the desired resolution of the cell subpopulation.

In an application where the number of cell ploidies is to be measured, cells within a sample may be stained with a DNA dye and the density of the stain is then quantitatively analyzed (where "density of stain" refers to the optical signal density of the dye). The dye signal density of such stains depends on the DNA/dye ratio (that is, the ratio of the amount of DNA stained by the dye to the total amount of dye added). If a predetermined amount of dye is added to each sample, the high cell concentration sample will be less bright than the low cell concentration sample, regardless of the sample characteristics. This situation would confound the quantitative analysis of the DNA content in each cell. As shown herein, estimating the number of nucleated cells in a sample prior to dye addition allows us to adjust the amount of dye used to quantify the DNA and DNA content of each cell in the sample. Thus, for example, a sample or a dispensed sample may be treated with a stain or dye that is directed to a cell surface marker representing the measured amount of cells, and the surface marker used to provide a non-destructive estimate of the concentration of cells within the sample. This estimated concentration may be used to calculate the amount of dye that needs to be added to the sample in order to always maintain a constant DNA-dye ratio (mole: mole) for subsequent measurements.

In a first demonstration of counting cells using a fluorescence-based method, a method may include determining the number of ploidies of the cells (e.g., counting cells by staining with a fluorescent conjugated antibody). In this non-limiting example, it is desirable to count WBCs in a blood sample so that a given number of WBCs can be stained with a predetermined concentration of DNA dye (e.g., 4' -6-diamidino-2-phenylindole (DAPI), or 1, 5-diamino-4, 8-dihydroxy-9, 10-anthracenedione)Or propidium iodide, or other DNA dyes). The method in this example involves counting WBCs using a fluorescent conjugated antibody and a spectrophotometer. It will be appreciated that this method may be helpful when using a DNA dye to stain cells and determine the ploidy number, where a ratio of the number of cells to the concentration of DNA dye (cell #: DNA dye) is required]) To generate comparable and stable data. Given the variation in the number of cells per milliliter of whole blood in healthy populations, there is a clear need to determine the number of WBCs per milliliter of blood before attempting to perform a ploidy stain.

In one example, the procedure involves first staining the cells with a fluorescent conjugated antibody (preferably an antibody directed against a broadly expressed antigen, such as CD 45; or an antibody specific for a subpopulation of cells, such as CD3 directed against T cells), or a fluorochrome dye (e.g., a cell membrane or cytoplasmic stain such as eosin, or a hemagglutinin or other stain or dye) that can label all cells, when the fluorescence wavelength from the fluorophore is spectrally distinguishable from the radiation wavelength of the DNA dye (preferably a wavelength at some distance therebetween). After a period of incubation, the sample is washed to remove excess (unbound) antibody, prepared in the appropriate volume and analyzed by a spectrophotometer. The resulting data allows the determination of the number of WBCs in a blood sample so that a specific volume of blood sample can be dispensed (to create a specific/desired number of WBCs) and stained with DNA stain. The resulting data is useful in calculating and adjusting the amount of DNA dye used to stain a specimen using the described fluorophore conjugated antibodies based on the determined WBCs amounts.

A further example comprises determining the number of cells (by DNA staining) prior to cell surface staining. Additional description may also be found in the cell count section below. It is sometimes desirable to count WBCs within a blood sample so that a given number of WBCs can be stained with an optimal concentration of antibody. In one example, the method comprises counting WBCs using a DNA dye and a spectrophotometer, as described above.

Alternatively, if the number of cells per ml prior to staining has been determined, a known number of cells can be aliquoted and each sample aliquoted stained regardless of (i) variation in healthy population and (ii) disease status. In order to determine the number of cells per ml of blood, it may be necessary to use DNA dyes, such as DAPI,Or propidium iodide. Alternatively, unbound dye may be eluted. A spectrophotometer can be used to determine nucleated cells per milliliter (e.g., per milliliter) of bloodPositive) number.

The number and concentration of White Blood Cells (WBCs) in an aliquot of blood sample may vary from subject to subject. However, in order to properly analyze the WBCs in a blood sample, it may be necessary to add a sufficient amount of a reagent (e.g., an antibody to a target specific WBC-specific antigen) depending on the number and concentration of WBCs in the blood sample. A process known as "dynamic dilution" may be used to ensure that sufficient antibody reagent is added to a sample. In a non-limiting example, the procedure is used to process blood cells to obtain a temporary cell count for determining the correct dosage of a reagent (e.g., a reagent for staining White Blood Cells (WBCs)) Antibody mixtures were used with the sample to provide complete cell staining). During this procedure, the cell is stained with a DNA dye (e.g., DAPI,Or propidium iodide) from the wavelength spectrum of the radiation of the fluorophore-conjugated antibody used in the subsequent steps or assays. Alternatively, after a period of incubation, the sample may be washed to remove excess (unbound) DNA dye. After a period of incubation, the sample may be prepared in suitable volumes and imaged or measured using a spectrophotometer. The resulting data allows the count or determination of the number of WBCs in a sample of known volume so that a given volume of blood sample can be dispensed (resulting in a particular/desired number of WBCs) and stained with the correct amount of antibody (e.g., DNA dye used is determined based on the estimated number of WBCs, and the desired antibody dose may be determined to provide antibody staining of the desired saturation). Thus, the estimate provided by DNA staining allows us to calculate and add the correct amount of antibody required for the number of WBCs in the dispensed sample.

Dynamic dilution operating specification:

in one example, a dynamic dilution protocol involves obtaining a aliquot of blood containing white blood cells to estimate the reagent dose containing the target WBCs antibodies, which is necessary for sample analysis.

In this non-limiting example, a blood sample of known volume is taken. A known amount of a nuclear dye (e.g. analogous to propidium iodide, DAPI, orA DNA-staining dye) is added to this known volume of sample. The mixture is incubated at a temperature of 25 ℃ to 40 ℃ for 2 to 10 minutes.

Next, a Red Blood Cell (RBC) lysis buffer was added. In this non-limiting example, the mixture is incubated at a temperature of 25 ℃ to 40 ℃ (lower temperatures may also be used) for 2 to 10 minutes. A suitable lysis buffer may be, for example, a hypotonic physiological saline solution; a low-profile sucrose solution; an isotonic ammonium chloride solution; an isotonic solution comprising a mild surface active substance like saponin; or other buffer in which RBCs can lyse. In an example, the lysis buffer includes a fixative such as paraformaldehyde to help stabilize the WBCs. A surfactant such as saponin can cause a large number of pores in the cell membrane. Red blood cells, due to their unique membrane properties, are particularly sensitive to such pore formation and are completely lysed, with the contents leaking into the surrounding fluid. Fixatives are added to prevent unnecessary lysis of the leukocytes. Platelets also remain unlysed. The purpose of this step is to remove red blood cells from the mixture, since they far exceed the number of white blood cells in a ratio of about 1000: 1. Platelets have no effect on imaging and are therefore not a factor considered in this process. In examples, a lysis buffer may also contain a known concentration of non-fluorescent beads; these beads can be used as markers for size and/or concentration. RBCs lysis, in conjunction with steps subsequent to this operating specification, can substantially remove any interference from RBCs with imaging or WBCs optical measurements.

The processed sample is then separated, wherein the separation step may be performed by any suitable method, such as, but not limited to, centrifuging the processed sample at 1200Xg for 3 minutes in a centrifuge.

After separation (e.g., centrifugation) the supernatant is removed; the remaining cell pellet was resuspended. In an example, the cell pellet is resuspended in some or all of the supernatant. A known volume of solution containing the resuspended cell mass comes from this step.

If desired, a further separation step and a further resuspension step may be carried out. These steps provided a concentrated sample with approximately 10-fold concentration of cells (ignoring any possible cell loss in each step).

The DNA stain dose in the resuspended and concentrated sample is then measured.For example, from a source such asThe fluorescence of the fluorescent DNA staining dye may be measured in a spectrophotometer. In an example, the sample may be measured at a wavelength of 632nm (C:)Activation wavelength) of light, the light waves emitted by the cell suspension may be filtered by a 650nm long pass filter, and the radiated light waves may then be measured in a spectrophotometer. This measured radiowave is correlated with a previously generated calibration curve to estimate an approximate concentration of leukocytes in the cell suspension. Typically, the cell concentration is in the range of about 1000 cells/ml to 100000 cells/ml. The amount of WBCs present in the sample estimated in this manner may be used to calculate a proper dilution factor for subsequent quantitative measurements to ensure that the cell concentration of the sample is limited to a predetermined target concentration range (e.g., a double or other range). The sample is then diluted according to the calculated dilution factor to provide a sample having a concentration of WBCs within a desired concentration range.

The purpose of this "dynamic dilution" step is to ensure that the concentration of WBCs contained in the sample is not too high or too low. If the cell concentration is too high, the accuracy of image processing operation is impaired; at too low a concentration, the number of cells in the sample may be insufficient. A concentrated sample dilution as shown herein can provide a desired range of WBCs concentrations and ensure that the signal from the sample during analysis is within an optimal range for detection and analysis.

Moreover, the number of WBCs estimated in this way allows us to calculate the required reagent dose for further examination of the sample and method steps; since the number of WBCs in a sample may vary, the required dose of reagents for each assay may depend on the number of WBCs in the sample being assayed. For example, the reagents added after estimation of the number of WBCs by the dynamic dilution protocol include antibodies to antigens specific to different types of WBCs, or, if such antigens are found in multiple types of WBCs, the amount of such antigens expressed in the different types of WBCs may vary. In the absence of such an estimate of the number of WBCs in a sample, predetermined doses of dye and other reagents must be added in subsequent tests of the sample, which can result in incorrect reagent doses and inaccurate or incomplete test results. Thus, the dynamic dilution protocol provided is a very important and useful initial step in the overall testing of a blood sample from a patient, allowing us to make more accurate and precise measurements of the sample.

Dynamic dyeing

In some examples, methods, systems, and apparatus are provided herein for dynamic staining of cell-containing samples.

Measurement of a component of interest in cells of a cell population

In one example, a method of dynamically staining a cell sample is associated with a method of measuring a component of interest in a cell of a population of cells.

As used herein, a "component of interest" refers to any type of molecule that may be expressed in a cell. "Components of interest" include proteins, carbohydrates, and nucleic acids. Typically, a "component of interest" is a particular type of molecule, such as a particular antigen. A non-limiting "component of interest" of a cell includes: CD5 protein, CD3 protein, and the like.

As used herein, a "cell population" refers to any group of cells based on one or more common characteristics. A "cell population" may have any degree of coverage, and may include a large number of cells or a small number of cells. Non-limiting examples of "cell populations" include: red Blood Cells (RBCs), White Blood Cells (WBCs), B-cells, CD34+ B-cells, and the like.

In some cases, it may be desirable to quantitatively measure a component of interest in cells of a particular cell population in a sample from a subject. For example, it may be desirable to measure the degree of CD5 ("component of interest") expression of B-cells ("cell population") in a sample from a patient with chronic lymphocytic leukemia. Detection and/or measurement of the level of a component of interest may involve the use of a binder molecule, such as an antibody or single chain variable fragment ("scFv"), that has an affinity for the particular component of interest. In order to accurately measure the level of a particular component of interest in a cell by a method involving the use of a binder molecule, it may be advantageous to expose the cell to a proportion or range of proportions of the binder molecule for the target component of interest. For example, it may be desirable to provide a dose of the conjugate to a sample of cells such that a linear relationship is formed between the dose of the component of interest in the cells and the dose of the conjugate that binds to the component of interest in the cells. For example, it may be unnecessary to dose the binder too little (so that not enough binder binds all of the components of interest in the cell) or too much (so that the binder binds non-specifically to the cell).

It may be difficult to provide a sample with an appropriate level of binding partner to accurately measure the amount of a component of interest in a population of cells in the sample using conventional methods, based on the fact that the size of the population of cells and/or the component of interest in the sample may vary from sample to sample. Rather, provided herein are methods, apparatus and systems for dynamically staining a cellular sample to accommodate samples containing a wide range of cell populations and components of interest.

In one embodiment, a method for measuring a component of interest in a cell of a population of cells in a sample is provided. The method is not limited, but may include one or more of the following steps:

first, a quantitative or semi-quantitative measure of the expression of a marker in the cells of the cell population may be obtained. The marker may be any marker that is expressed in the cell population of interest, and it may be a marker that is uniquely expressed in the cell population of interest (e.g., not expressed in any other cell type in the sample). The marker may be measured by any method that does not disrupt the sample and may use any system or device. A conjugate that identifies the marker may be mixed with the sample. The binding entity may contain an attachment molecule to facilitate detection of the binding entity (e.g., a fluorescent label). In one example, the marker may be detected and/or measured by a fluorescence spectrophotometer. In one embodiment the conjugate has a fluorescent label, the label is measured by a fluorescence spectrophotometer; a spectrofluorometer may be used to measure a substantial amount or fraction of the fluorescent signal from the sample, rather than measuring the fluorescent signal from a single cell.

Second, based on a quantitative or semi-quantitative measurement of expression in the cells of the cell population, an approximate number and concentration of cells of the cell population in the sample may be determined. An approximate number and concentration of cells of the cell population in the sample may be determined, for example, by applying a calibration curve. Calibration curves may be plotted and/or may be provided for different combinations of labels/conjugates. The calibration curve may be plotted, for example, by measuring signals from a known number of cells that contain a certain label and bind to a certain binding partner. In some instances, an approximate number and concentration of cells of the cell population in the sample may be determined with the aid of a computer. In some aspects, an approximate number and concentration of cells of the population of cells in the sample may be determined that is no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500% of the true concentration.

Third, depending on the determined number or concentration of cells in the cell population in the sample, an amount of a reagent added to the sample may be selected, wherein the reagent specifically binds to the component of interest in the cells of the cell population. The reactant may be, or include, any molecule that specifically binds to the component of interest. For example, the reagent may be a binding body, such as an antibody. The reagent may be designed to be detectable at any time (e.g., by fluorescence or luminescence) and/or, at least in some cases, to produce a detectable signal. In some examples, the reagent may be attached to a molecule to aid in the detection of the reagent. The dose of reagent added to the sample may be any dose. In some instances, a reagent dose may be added to the sample such that a linear relationship is formed between the level of the component of interest in the cell and the signal produced by the conjugate that binds to the component of interest in the cell.

Fourth, after a dose of a reagent added to the sample is selected, the selected dose of reagent may be added to the sample.

Fifth, because the reagent binds to the component of interest, cells in the sample may be tested.

Sixth, the amount of the component of interest in the cells of the sample population may be determined based on the dose of the reagent that binds to the component of interest.

In some examples, the fifth and sixth steps may be performed together such that the measurement of the amount of the reagent bound to the component of interest is sufficient to confirm the amount of the component of interest in the cells of the sample population.

In other examples, provided herein are systems and devices for dynamic staining of samples. The system and apparatus may include, but are not limited to, a spectrophotometer and a fluorescence microscope. In one example, a system or method for dynamic staining of a sample may be designed as described in U.S. patent application No. 13/355,458, which is incorporated by reference in its entirety. In one example, the system and apparatus may automatically determine the amount of a reagent added to a sample to determine the amount of a component of interest in cells of a population of cells of the sample based on a measurement of the amount of a marker expressed in the cells of the population. In another example, the system and apparatus may automatically determine the amount of a reagent added to a sample to determine the amount of a first component in cells of a sample population of cells based on a measurement of the amount of a second component in the cells of the sample population.

Ambient based auto-focus

In some examples, methods, systems, and apparatus are provided herein for ambient-based microscope autofocus.

Many clinically relevant substances in biological samples span a wide range of sizes (e.g., length, height, or other measurements). For example, bacteria are typically about 1 μm long, red blood cells about 6-8 μm long, white blood cells about 10-12 μm long, epithelial cells may be about 100 μm long, and casts and crystals may be about 200-300 μm long. In addition, there are some intangible elements, such as urinary tract mucus, which is usually present in a thread or thread form, with a possible length range of about 10-400 μm.

One challenge of microscopic imaging techniques is to acquire accurate images of a field of view containing an unknown and randomly composed object of different sizes, such as those described above. Since the depth of focus of many microscope objectives is limited (typically about 1-10 μm), for a given field of view containing elements of different sizes, it may be necessary to acquire multiple focal planes for that field of view in order to obtain accurate, sharp images of the different elements within that field of view. One problem with many conventional auto-focusing methods is that they are designed to focus on a major structure within a field of view so that the graphic clarity of that structure can be maximized. This approach may be ineffective for taking elements of different sizes within a sample.

In one example, a method for ambient-based microscope autofocus is provided that includes mixing a reference particle of known size with a sample for microscopic imaging. In an example, more than one reference particle is added to the sample; preferably or substantially all such reference particles are of the same known size. In the example, the number of reference particles added to a particular sample volume is known. The reference particle may be detected during microscopic imaging and used to achieve a focusing effect. When the focusing effect is achieved by using the reference particles, it is possible to select a focusing plane that is independent of the overall image composition. In one aspect, the method may be useful in focusing a sample containing unknown constituent elements. On the other hand, the method may support the generation of an accurate focal plane regardless of the accuracy of the microscope or microscope-related hardware. For example, when a focal plane is selected based on feedback of the sharpness of the reference particles in the field of view, it is possible to achieve accurate focusing of different elements in a sample, regardless of the level of accuracy and precision of the focusing hardware [ e.g., microscope objective, sample holder shape (e.g., a cuvette or slice), or a sample holder with non-uniformity ].

In one example, a reference particle may contain or be labeled with a molecule to aid in detecting the particle during microscopic imaging. In one example, a reference particle may be labeled with or contain a fluorescent molecule. The fluorescent molecule may absorb a light wave of a first wavelength and then, in response to absorbing the light wave of the first wavelength, it may release a light wave of a second wavelength. In one example, a sample mixed with a reference particle may be exposed to a wavelength of light that activates a fluorescent molecule in a reference particle of interest, and the light emitted from the fluorescent molecule may be measured. Specific fluorescence from a reference particle may be used to detect the reference particle, and information detected from a sample reference particle may be used for auto-focus.

The reference particle may be of any shape, for example spherical or cubic. Reference particles include, but are not limited to, beads and microspheres. The reference particle may be of any size, such as approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 μm in diameter or length. The reference particle may be made of or contain any suitable material, such as polystyrene, polyethylene, latex, acrylic plastic, or glass. For example, a reference particle may be a polystyrene bead, such as a polystyrene bead having a diameter of between about 0.1 μm and 50 μm; or between about 1 μm and 20 μm; or between about 5 μm and 15 μm; or about 10 μm in diameter.

In one example, a method for focusing a microscope is provided, which may include one or more of the following steps. First, a sample containing the target (e.g., bacteria, red blood cells, etc.) to be microscopically analyzed may be mixed with a reference particle. The reference particle may contain or be labelled with a molecule to aid detection of the particle during microscopic imaging, for example a fluorophore. Second, the mixture containing the reference particles and the sample may be placed in the optical path of a microscope, for example in a cuvette or slide. Alternatively, the reference particle may be deposited to the bottom of the sample in the cuvette or slice, that is to say the reference particle falls on the lowermost surface of the cuvette or slice in contact with the sample. The microscope may be of any type, including a fluorescence microscope. Third, the mixture may be exposed to a light wave designed to cause the reference particle to be seen. The beam may be of any type and may come from any direction relative to the reference particle. For example, the beam may be at a wavelength that activates a fluorophore located within or attached to the reference particle. Exposing the reference particle to the light wave may result, for example, in the generation and release of light waves at a particular wavelength from the reference particle, and/or scattered light waves from the reference particle. Fourth, the emitted or scattered light waves from the reference particle may be detected by the microscope, and this information may be used to determine the position of the reference particle within the mixture and/or to focus the microscope. Alternatively, the microscope may be focused to a focal plane that is appropriate for an object of the same size as the reference particle. An image from the microscope may be obtained by an image sensor. The image may be saved and used for image analysis.

In some instances, multiple reference particles may be added to a sample. The reference particles may be the same size or may be different sizes. In some examples, reference particles of different sizes contain different fluorophores. Different fluorophores may contain different absorption wavelengths, different emission wavelengths, or both.

In one example, a method for focusing a microscope is provided that includes mixing more than one known size of reference particles with a sample for microscopic analysis, wherein at least two of the reference particles have different sizes and contain different fluorophores. The method may include one or more of the following steps: first, it is possible to mix a sample containing a target for microscopic analysis with more than two reference particles, at least two of which possess different sizes and contain different fluorophores (e.g., "first reference particle" and "second reference particle"). Second, the mixture containing the reference particles and the sample may be placed in the optical path of a microscope. The microscope may be of any type, including a fluorescence microscope. Third, the mixture may be exposed to a light wave designed to cause the first reference particle to be seen. The beam may be of any type and may come from any one direction relative to the first reference particle. For example, the beam may be at a wavelength that activates a fluorophore located within or attached to the first reference particle. Exposing the first reference particle to the light beam may cause generation, release, or scattering of light waves at a particular wavelength from the first reference particle. Fourth, the radiated or scattered light waves from the first reference particle may be detected, and this information may be used to determine the position of the first reference particle within the mixture and/or used to focus the microscope to a first plane for application to objects of similar size to the first reference particle. Alternatively, an image from the first focal plane may be obtained by an image sensor. The image may be saved and used for image analysis. Fifth, the mixture may be exposed to a light wave designed to cause the second reference particle to be seen. The beam may be of any type and may come from any one direction relative to the second reference particle. Exposing the second reference particle to the light beam may cause generation, release, or scattering of light waves at a particular wavelength from the second reference particle. Sixth, the radiated or scattered light waves from the second reference particle may be detected, and this information may be used to determine the location of the second reference particle within the mixture and/or used to focus the microscope to a second plane for application to objects of similar size to the second reference particle. Alternatively, an image from the second focal plane may be obtained by an image sensor. The image may be saved and used for image analysis.

In other examples, provided herein are systems and apparatus for ambient-based microscope autofocus. The system and apparatus may include, but are not limited to, a fluorescence microscope. In one example, the system and apparatus may automatically add a reference particle of known size to a sample for microscopic analysis to form a mixture; placing the mixture in the optical path of a microscope; exposing the mixture to a light beam to make the reference particle visible; the position of the reference particle in the mixture is determined and the microscope is focused according to the position of the reference particle in the mixture. In one example, a system or method for ambient-based microscope autofocus may be designed as described in U.S. patent application No. 13/355,458, which is incorporated by reference herein in its entirety.

Positioning a sample holder

In some examples, methods, systems, and apparatus are provided herein for determining the position of a sample rack, a portion of a sample rack, or an indicator thereon. Such a determination is preferably an accurate determination useful for identifying cells, particles or other substances within sample holders located in a field of view even after a sample holder has been moved or a field of view has been changed (e.g., by changing the focal length, or by viewing different areas within a sample holder).

In an example, an image based feedback mechanism may be used to accurately and precisely determine a particular location in a cuvette, such as in a tube or other sample-containing region (see an analysis region 608 shown in fig. 7 and 8). This determination is important for comparing the images and optical measurements taken before and after the movement, particularly when the sample holder is moved and then returned to its original position. Variability from different sources may affect the position of the sample relative to the imaging system pivot; for example, variability in cuvette parts, variability in cuvette combinations, variability in cuvette position in the imaging system, and other possible sources of variability may affect the position of a sample relative to the imaging system even though the sample remains in the same position throughout the sample holder. Methods for identifying and describing the position of a sample holder relative to an imaging system are shown herein. For example, a cuvette registration program may be run in order to accurately and reproducibly image a region of interest within a cuvette. In an example, such a procedure begins with the analysis of an image taken at a predefined location in a sample holder that is in close proximity to a registration structure or fiducial marker within the field of view, or a location that can be detected by the procedure. A cuvette registration program contains an image processing program that looks up the presence of a fiducial marker in an image and replies with a yes/no answer (depending on whether the fiducial marker is found in a particular region) or the likelihood that the marker appears in the image. In the demonstration, the fiducial identification is not found within the queried region, and then a query rule is used, i.e., the queried region is moved to a different location or re-imaged within the sample holder. This operation is repeated until the program finds the reference identifier (e.g., a "yes" answer is made to the question of whether the reference identifier is found within the query, or the identifier is most likely in that area). Once the fiducial marker location is determined, all other locations within or on the sample rack can be determined since the size and layout of the sample rack is known. In this way, after the location of the fiducial marker is determined, any points of interest used for imaging may be found and imaged because their locations are also known (e.g., their distance and orientation from the fiducial marker is known, and because the location of the fiducial marker is known, the location of the points of interest is also known). In examples, a fiducial mark may be, or include, a specifically designed structure (e.g., perhaps a hole, a protrusion, a pattern of stamped or molded, or other structure) located on the cuvette that may be molded in the same location with any one of the desired tolerances for each of the portions. In an example, a fiducial mark may be, or include, a structure of a cuvette (e.g., the edge of a tube) that is always present at a fixed distance from the point of interest (e.g., the fiducial mark is disposed on the edge of the tube, and the fiducial mark is always a fixed distance from the central axis of the tube).

Cell count/count cells

In some examples, methods, systems, and apparatus are provided herein for counting cells within a sample.

A particular conventional method of staining a cell-containing sample involves staining a particular volume of the sample (e.g., blood) with a concentration or amount of a staining agent. This may be referred to as "volume staining". Volume staining has a number of disadvantages including: (i) it does not elucidate the normal variation of cell subsets between different subjects (different numbers of cell subsets may vary greatly between different healthy subjects, such as CD3+ T cells (CD3+ indicates that T cells express the CD3 marker)); (ii) it does not demonstrate that pathological samples may contain very different cellular components than normal samples (e.g., the percentage and number of CD3+ T cells in blood samples is significantly higher in T cell leukemia patients than in normal subjects).

In order to accurately and reproducibly stain a cell-containing sample, it may be necessary to add a specified amount of cell stain (e.g., DNA dye, antibody, conjugate, etc.) to a specified amount or concentration of cells. For example, it may be desirable to add 0.2 micrograms of a particular stain to every 1000 leukocytes in a sample. After incubating the dye with the cells for a period of time, the sample may be washed to remove excess (unbound) dye, prepared to an appropriate cell density and imaged. In this method, a staining agent and staining process can be optimized or normalized for a particular cell number.

In one example, a method is provided for counting the number of cells of interest within a sample. The method may include one or more of the following steps or elements: a first stain that binds to cells of interest within a sample may be added to the sample. The mixture of the first stain and the sample may be incubated. The first stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained with the first stain may be prepared to a desired volume for further analysis. The washed cells stained with the first stain may be analyzed by a spectrophotometer. Data from the spectrophotometer may be used to count the approximate number of cells within the sample. For example, the first stain may be a fluorescent dye associated with the cell nucleus, and the spectrophotometer may include a light source that emits light at the activation wavelength of the fluorescent dye and a light sensor that detects light at the emission wavelength of the fluorescent dye. In this example, based on the fluorescence signal of the dye, the approximate number of nucleic acids in the sample may be calculated; and the approximate number of cells in the sample can be determined from the approximate number of nucleic acids in the sample. Depending on the approximate number of cells within the sample, a second stain may be added to the sample that binds to the cells of interest within the sample. In an example, the dose of the second stain added to the sample may be determined by the approximate number of cells determined by the first stain. In an example, the dose of the second stain added to the sample may be calculated by using the number of cells determined by the first stain to obtain an ideal ratio of the second stain to each cell. The mixture of the second stain and the sample may be incubated. The second stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained with the second stain may be prepared to a desired volume for further analysis. The washed cells stained with the second stain may be analyzed by a microscope.

Counting the number of cells in a sample prior to determining cell ploidy

In one example, a method for counting the number of cells within a sample prior to determining cell ploidy is provided, wherein the method comprises one or more of the following steps or elements. A first stain that binds to cells of interest within the sample and has spectral characteristics different from a DNA dye emission wave may be added to the sample. The cell of interest may be, for example, a leukocyte. The first staining agent may be, for example, a fluorophore conjugated antibody. The fluorophore conjugated antibody may be conjugated to, for example, a broadly expressed antigen (e.g., CD45), or may be conjugated to an antigen expressed by a specific subset of cells (e.g., CD3 of T cells). The mixture of the first stain and the sample may be incubated. The first stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained by the first stain may be prepared to a desired volume for further analysis. The washed cells stained with the first stain may be analyzed by a spectrophotometer. Data from the spectrophotometer may be used to count the approximate number of cells within the sample. Depending on the approximate number of cells within the sample, a second stain that binds to cells of interest within a sample may be added to the sample. The second stain may be a DNA dye such as propidium iodide or 4',6 diamidino-2-phenylindole hydrochloride ("DAPI"). In an example, the dose of the second stain added to the sample may be determined by the approximate number of cells determined by the first stain. In an example, the dose of the second stain added to the sample may be calculated by using the number of cells determined by the first stain to obtain an ideal ratio of the second stain to each cell. The mixture of the second stain and the sample may be incubated. The second stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained by the second stain may be prepared to a desired volume for further analysis. The washed cells stained with a second stain may be analyzed for ploidy by a microscope.

In methods for determining the ploidy of a cell, it may be important to combine a given number of cells for ploidy analysis with a certain amount or concentration of DNA stain in order to generate accurate and consistent data regarding the ploidy of the cell. In one example, the number of leukocytes per milliliter of blood in a healthy population may vary, and thus the number of leukocytes in a volume of blood may need to be determined before attempting a ploidy analysis of the stained leukocytes.

The methods provided above for analyzing the ploidy of a cell may also be used as any method that requires counting the number of cells in a sample prior to determining a nucleic acid content-related characteristic of a cell. For example, the method may be used with a method of counting cells in a sample prior to determining the morphology of the nuclei, the size of the nuclei, the ratio of the area of the nuclei to the area of the whole cell, and the like.

Counting cells in a sample prior to cell surface staining

In one example, a method for counting the number of cells within a sample prior to cell surface staining is provided, wherein the method comprises one or more of the following steps or elements. A first dye that binds to a cell of interest in the sample and has a spectral characteristic different from a dye emission wave used to stain the surface of the cell A color agent may be added to the sample. The cell of interest may be, for example, a leukocyte. The first dye may be, for example, a DNA dye (e.g., propidium iodide, ethyl acetate,Or DAPI). The mixture of the first stain and the sample may be incubated. The first stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained by the first stain may be prepared to a desired volume for further analysis. The washed cells stained with the first stain may be analyzed by a spectrophotometer. Data from the spectrophotometer may be used to count the approximate number of cells within the sample. Depending on the approximate number of cells within the sample, a second stain that binds to cells of interest within a sample may be added to the sample. In an example, the dose of the second stain added to the sample may be determined by the approximate number of cells determined by the first stain. In an example, the dose of the second stain added to the sample may be calculated by using the number of cells determined by the first stain to obtain an ideal ratio of the second stain to each cell. The second staining agent may be, for example, a fluorophore conjugated antibody. A fluorophore conjugated antibody may be conjugated to, for example, a broadly expressed antigen (e.g., CD45), or may be conjugated to an antigen expressed by a specific subset of cells (e.g., CD3 of T cells). The mixture of the second stain and the sample may be incubated. The second stain and the cells in the sample mixture may be washed to remove excess (unbound) stain. The cells stained by the second stain may be prepared to a desired volume for further analysis. The washed cells stained with a second stain may be analyzed by a microscope for a cell surface antigen.

In methods of cell surface antigen staining, it may be important to combine a given number of cells for analysis with a certain amount or concentration of cell surface antigen stain in order to generate accurate and consistent data regarding the amount of the cell surface antigen. In one example, the number of leukocytes per ml of blood in a healthy population may vary (typical WBC content of 3000-10000 per μ L in blood from healthy subjects), and thus the number of leukocytes within a certain blood volume may need to be determined before attempting to stain the leukocytes for cell surface antigen analysis. In another example, the number of leukocytes per milliliter of blood may vary between healthy subjects and diseased individuals (e.g., a lymphoma patient may have up to 100,000 WBC levels per μ L of blood), and thus the number of leukocytes within a certain blood volume may need to be determined before attempting to stain the leukocytes for cell surface antigen analysis.

Thus, as a theoretical demonstration, a healthy subject may contain 5000 cells per microliter of blood, 500 of which are CD3 +; while a lymphoma patient may contain 50000 cells per microliter of blood, 45,000 of which are CD3+ T cells. If 100 microliters of blood is routinely stained, a sample from a healthy subject will stain approximately 500,000 cells, of which approximately 50,000 cells are CD3+ T cells. One 100 microliter of blood from a lymphoma patient contains about 5,000,000 cells, of which about 4,500,000 are CD3+ T cells. In this theoretical demonstration, the pathological sample contained a total of ten times the number of cells and ninety times the number of T cells of CD3+ in a sample from a healthy subject. If the pathological sample is stained using the conventional optimal "volume staining" method from healthy subjects, the sample from the lymphoma patient may be insufficiently stained. For this reason, the current method is, by way of example, superior to conventional volumetric staining methods, wherein a pre-estimated number of cells within a sample is used to adjust the amount of dye applied to the sample.

In view of this, the methods provided herein may be used to count the number of cells within a sample prior to cell staining in order to generate accurate and/or consistent data about the sample.

Acceleration method

Some of the methods, systems, and devices provided herein may support rapid acquisition of sample analysis results. The methods provided herein may take less than, for example, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes from the start of the method to the time at which the results of the analysis are provided.

The results of the rapid analysis may be used to provide real-time information relating to the treatment, diagnosis or monitoring of a patient. For example, the results of the rapid analysis may be used to guide a processing decision of a surgeon performing a procedure on a patient. In the middle of a procedure, a surgeon may obtain a biological specimen from a patient for analysis. With one method provided herein, a surgeon, upon receiving a rapid analysis of the sample, may be able to make a processing decision during the procedure.

In another example, the rapid analysis results provided by the methods, systems, and apparatus provided herein may support a patient receiving information about a biological sample provided by the patient during the same visit at the same point-of-care service.

For example, using a rapid assay as described herein, it may be used to prepare a whole blood sample for analysis of multiple marker expression and cell types in leukocytes. Such an assay is also very useful for preparing a whole blood sample for imaging analysis; the sample preparation for imaging does not exceed 20 minutes, or does not exceed 15 minutes.

Rapid leukocyte detection from whole blood samples

This assay prepares a whole blood sample for cellular analysis of leukocytes for no more than 15 minutes, or no more than 20 minutes. Automated cell analysis of such prepared cells may be accomplished quickly, and therefore WBC cell analysis of whole blood may be accomplished in about a half hour or less. Moreover, such tests use only small volumes of blood samples, which are not disturbed in their source and are less inconvenient and uncomfortable for the patient than tests requiring large volumes of blood samples.

The reagents used in this assay include: phosphate buffered saline, lysis fixation buffer, beads, resuspension buffer, and a mixture of reagents containing a dye and a dye-conjugated antibody. The antibody is directed to WBC specific markers.

Phosphate Buffered Saline (PBS): 137mM NaCl,3mM KCl,8mM, Na2HPO4,1.5mM KH2PO4, pH adjusted to pH 7.2-pH 7.4 (with HCl).

Resuspension buffer (RSB): PBS containing 5% calf serum protein.

Lysis of the fixation buffer: PBS containing 0.0266% saponin and 10% Paraformaldehyde (PFA), wherein "%" is indicated as grams/100 mL (final ratio approximately 13:1 saponin PBS: PFA).

Reaction reagent mixture 1:and Pacific Blue^TMDye conjugated anti-CD 14 antibody, Fc blocking antibody (e.g., an immunoglobulin such as mouse IgG), PBS containing 0.2% BSA.

Reaction reagent mixture 2: anti-CD 16 antibody conjugated with PE dye, Alexa647 dye-conjugated anti-CD 45 antibody, anti-CD 123 antibody conjugated with PECy5 dye, Fc blocking antibody (e.g., an immunoglobulin), PBS containing 15% BSA.

The detection step comprises:

whole blood is obtained from a subject.

50 μ L of whole blood was placed in the tube. The whole blood sample may be collected directly from a tube, if desired. If the total volume of whole blood collected from the subject is 50 μ L, then the entire sample is added or collected into a tube; if the sample collected from a subject is greater than 50 μ L, the sample is dispensed at 50 μ L.

The sample was centrifuged at 1200Xg for 3 minutes.

Remove 20 μ L of plasma from the tube.

The tube was placed on a hot plate (warmed to 37 ℃ C.), 20. mu. LRSB was added thereto, and mixed well.

Mix 1 (approximately 5 μ L) was added. (in the example, mixture 1 may be added directly to whole blood, and the preceding centrifugation, removal of plasma, and RSB may be omitted during the suspension step.)

The samples were incubated at 37 ℃ for 2 minutes.

Lysis immobilization buffer was added (6: 1 ratio of lysis immobilization buffer to stained blood; approximately 300-. A known concentration of beads may be included in the lysis buffer to provide focus targets (reference particles) and to provide a calibration for sample concentration (e.g., as described in the "ambient-based autofocus" section above). Polystyrene or other beads having a diameter of between about 1 micron and about 30 microns may be used. For example, 10 micron polystyrene beads with a concentration of 100-.

The lysis fixation buffer was incubated at 37 ℃ for 3 min; after about 1.5 minutes of buffer addition, the solution was mixed five times up and down with a pipette.

The sample mixture was centrifuged at 1200Xg for 3 minutes.

The supernatant was removed (approximately 350. mu.L). The supernatant is preserved, if necessary, to adjust the volume in a later step.

Mix 2 (approximately 15 μ L) was added to provide the final mixture.

The final mixture was added to a preheated imaging cuvette (37 ℃).

The cuvette was incubated at 37 ℃ for 5 minutes before imaging.

The sample is imaged.

Thus, the sample can be imaged in less than 15 minutes. In an example, some steps may be shortened (e.g., in another example, one centrifugation step or one incubation step may be shortened). Since the method provided above uses a mixture containing multiple dyes to prepare a sample, analysis of the expression of several cell type markers in the sample may be performed in a single field of view, with minimal duplication in an effort to provide an adequate image of the sample. These light scattering images of the same field of view also provide another analysis that may be effectively used for analysis of several image patterns of a sample without the need to obtain different samples or different fields of view. A reference particle containing a known size may further aid imaging by using auto-focus; furthermore, since the concentration of the reference particles is known, it allows independent measurements of sample dilution and cell concentration for each image.

Imaging of the prepared sample may also be very rapid, for example, in the order of 10 minutes (typically 2-12 minutes) by automated equipment having the structure described herein, such as that described in U.S. patent application 13/244, U.S. patent application 94713/769,779 and related applications. Thus, the entire analysis process in an example, including the preparation of a blood sample and imaging the prepared sample, may be completed in about 30 minutes or less.

Imaging and image analysis of the prepared sample is suitable for identifying different cell populations in whole blood WBCs according to the methods discussed above (and similar methods discussed below). By illuminating the sample with light of different wavelengths, this identification and quantitative analysis of the same sample is quickly accomplished, and the imaging results and light intensity are recorded and analyzed. Such methods are suitable for providing images and graphics, for example, as shown in fig. 9, 10 and 11, all prepared using the methods shown herein (e.g., the methods discussed above and below). The comparison shown in FIG. 12 illustrates that these methods are accurate and reliable and are in good agreement with other methods (e.g., analysis by the Abbott CELL-DYN Ruby System (Abbott Diagnostics, Lake Forest, Ill., USA)) and a control analyzer for comparison is shown in FIG. 12.

Analysis of pathological samples

Any of the methods provided herein may be used to analyze a pathology sample containing cells. If a pathology specimen is a tissue sample, the sample may be processed to separate cells in the tissue into individual cells, which are then analyzed by the methods provided herein.

Analysis of pathological samples by any of the methods provided herein may support rapid pathological analysis, and such integrated rapid pathological analysis may ultimately lead to a treatment decision for the patient.

Additional operations in reaction to the results of the analysis

In some instances, the devices and systems provided herein may be designed to trigger an additional operation as a reaction to a result obtained by an analytical method provided herein.

In one example, an apparatus or system may be programmed to provide a warning to a user when a result is outside an expected range. Such warnings may prompt a user or medical personnel to perform operations such as human analysis of the sample, inspection of the device or system to ensure proper functioning, and so forth.

In another example, a device or system may be programmed to automatically run one or more additional tests on a sample when a result is outside an expected range. In some instances, the devices and systems provided herein are capable of performing a plurality of different detections; and the devices or systems may run an additional test to confirm or further investigate a result generated by a method provided herein.

Analysis with non-specific dyes

A non-limiting example of accelerated imaging is the use of a "highlighted" format, where cells are labeled with very high concentrations of dye. In the present example, non-specific dyes are used that can be used to label DNA, cell membranes, or other parts of cells. This demonstration did not use antibody dyes directed to specific or rare proteins or other markers.

After the use of non-specific dyes, cellular information may be obtained without the need for a separation step (e.g., centrifugation or physical separation). Without such a separation step, we can more quickly image directly into the sample, for example, but not limited to, imaging a large area of cells, including a) non-target cells, such as Red Blood Cells (RBCs), and b) target cells or objects of interest, such as White Blood Cells (WBCs). Thus, in a non-limiting example of imaging one blood sample, five million RBCs and five thousand or other numbers of WBCs may be imaged at one time. The target cells may also be classified according to intracellular composition, such as, but not limited to, nuclear shape of a cell. In one example, a nuclear stain is used to stain nuclei within a sample, and based on the type and amount of stain a particular cell contains (e.g., the expression of nuclear staining, or the shape of a stained nucleus, or other characteristic), we can determine its cell type from the staining, even though the stain is non-specific. In another example, other internal morphologies within the cell (e.g., whether the cytoplasm has particles or other material, etc.) may be indicative or characteristic and used to determine and quantify cells within a sample. For a urine sample, any expressed nuclei and crystalline shapes within the sample can be used to confirm the sample and determine if any abnormalities are found. Thus, the use of non-specific dyes can be used to rapidly image cells and, if desired, to determine cells to a certain extent.

Analysis using multiple activation and/or detection channels

In more advanced cell detection examples, an additional activation and/or detection wavelength may be used when using an even smaller sample volume for cell analysis. For example, for the classification of WBCs, different cells, such as T cells, B cells, K cells and others, are counted when detecting a lymphocyte subset. In this case, we used two markers only to confirm that the cell is a lymphocyte. For further subtype classification of cells from a blood sample, for example, we can use two markers again. Thus, if our system can only detect two colors at the same time, the number of wavelengths used for analysis is insufficient.

In one example, we can split a sample into two parts and then use the different split parts of the sample to perform one combined imaging in one part of the system and another combined imaging in the other part. Unfortunately, this can result in a doubling of time and the required capacity of the sample. The more independent channels that are placed in a system, the fewer the number of sample portions and volumes that are used.

Demonstration of

Cell processing

In an example, it is often very useful to process biological samples for imaging, detection and analysis. For example, it is often useful to process biological samples containing cells for imaging, detection, and analysis.

Processing of a biological sample may include pre-processing (e.g., preparation of a sample for a subsequent processing or measurement), processing (e.g., changing a sample to be different from its original or previous state), and post-processing (e.g., cleaning all or a portion of a sample after a measurement or use). A biological sample may be divided into several portions, such as a aliquot of a blood or urine sample, or such as a tissue sample that is sliced, minced, or otherwise cut into two or more portions. Processing of a biological sample, such as a blood sample, may include mixing, stirring, sonication, homogenization, or other processing of a sample or sample portion. The processing of a biological sample, such as a blood sample, may include centrifugation of a sample or sample portion. Processing of a biological sample, such as a blood sample, may include providing time to separate or precipitate components in the sample, and may include filtering (e.g., flowing the sample or a portion of the sample through a filter). Processing of a biological sample, such as a blood sample, may include allowing or causing agglutination of a blood sample. Processing of a biological sample, such as a blood sample, may include concentrating the sample or a portion of the sample (by sedimentation or centrifugation of a blood sample or a tissue homogenization solution containing a tissue sample from a tissue sample) to provide a precipitate and a supernatant. Processing of a biological sample, such as a blood sample, may include diluting a portion of the sample. It may be a dilution of a sample or a portion of a sample, including a dilution of a precipitate or a supernatant from a sample. A biological sample may be diluted with water or with a physiological saline solution, such as a buffered saline solution. A biological sample may be diluted with a solution that may or may not include a fixative (e.g., formaldehyde, paraformaldehyde, or other agents that crosslink proteins). A biological sample may be diluted with a solution effective to create an osmotic gradient between the surrounding solution and the interior or inner portion of the cell, effectively changing the volume of the cell. For example, if the final solution concentration after dilution is lower than the effective concentration in the interior or inner part of the cell, the volume of such cells will increase (e.g., cells will develop edema). A biological sample may be diluted with a solution that may or may not contain an osmotic agent (such as glucose, sucrose, or other sugars; sodium, potassium, ammonium, or other salts; or other osmotically active components or constituents). In an example, an osmotic agent may be effective to maintain the integrity of cells within the sample, such as by stabilizing or reducing the potential osmotic gradient between the surrounding solution and the interior or inner portion of such cells. In an example, an osmotic agent may be effective to provide or increase an osmotic gradient between a surrounding solution and an interior or inside portion of such a cell effective to at least partially collapse the cell (the interior or inside portion of the cell being less concentrated than the surrounding solution); or effectively edema the cells (the inner or inner part of the cell is more concentrated than the surrounding solution).

A biological sample may be contacted with a solution containing a surfactant, which may disrupt the cell membranes within the sample, or have other effects on cell morphology. For example, contacting RBCs with a low concentration of surfactant causes the RBCs to lose their disk-like morphology and assume a more spherical-like morphology.

A biological sample may be stained; or several markers may be added to the sample; or the sample may be prepared for detection, observation or quantitative analysis of the sample, a portion of the sample, a component in the sample, or a portion of the cells or structures within the sample. For example, a biological sample may be contacted with a solution containing a dye. The dye may stain or make visible a cell, or a portion of a cell, or a substance or molecule associated with a type of cell within a sample. A dye may bind to or be altered by an element, compound, or other component in a sample; for example, a dye may change color, or change one of its properties, including its optical properties, in response to a change or difference in the pH of the solution in which it is placed; a dye may change color, or change one of its properties, including its optical properties, to an element or compound (e.g., sodium, calcium, CO) in solution in which it is placed ₂Glucose, or other ions, elements, or compounds) in response to changes or differences in concentration. For example, a biological sample may be contacted with a solution containing an antibody or antibody fragment. For example, a biological sample may be contacted with a solution containing particles. Particles added to a biological sample may be used as a standard (e.g., may be a size standard, the particle size or particle size distribution of which is known; or a concentration standard, the quantity, volume, or concentration of which is known); or may be used as a marker (e.g., may bind or adhere to a particular cell or cell type, a particular cellular marker or cellular component, or bind to all cells in a sample).

Cell counting involves observing and measuring cells, e.g. red blood cells, hematoxylinPlates, leukocytes, including qualitative and quantitative observations and measurements of cell number, cell type, cell surface markers, intracellular markers, and other characteristics of the cells of interest. When a biological sample comprises or is itself a blood sample, the sample may be divided into portions and may be diluted (e.g., to provide a larger volume for convenient processing; to change the density or concentration of cellular components within the sample to provide a desired dilution density, concentration, or number of cells or range thereof, etc.). The sample may be treated with a reagent that affects agglutination; or processed to concentrate or precipitate sample components (e.g., ethylenediaminetetraacetic acid (EDTA) or adding anti-heparin to the sample; or the sample may be centrifuged or the cells allowed to precipitate). A sample or portion of a sample may be treated to add dyes or other reactive reagents that may react with and label specific cells or cellular components. For example, dyes for marking the nucleus of the cell (e.g. hematoxylin dyes, cyanine dyes, such as The Draq dye of (d) and other dyes); dyes that label the cytoplasm (e.g., eosin dyes, also including fluorescent dyes and other dyes) may be used separately or together to aid in the observation, confirmation, and quantitative analysis of the cells. Some specific markers may also be used in cell counting, including specific antibodies and antibody fragments directed against cellular targets, such as cell surface proteins, intracellular proteins and components, and other targets.

Biological samples may be measured and analyzed by cytometry using optical methods including, for example, photodiode detectors, photomultiplier tubes, charge coupled devices, laser diodes, spectrophotometers, cameras, microscopes, or other devices capable of measuring light intensity (a single wavelength, multiple wavelengths, or wavelengths within a range, or ranges), forming images, or both. A field of view comprising a sample or a portion of a sample may be imaged, scanned, or both using the detector. A biological sample may be measured and analyzed by a cytometer prior to processing, dilution, separation, centrifugation, agglutination, or other alteration. A biological sample may be measured and analyzed by a flow cytometer between or after being processed, diluted, separated, centrifuged, agglutinated or otherwise altered. For example, a biological sample may be received and measured and analyzed directly by a flow cytometer. As another example, a biological sample may be measured and analyzed by a cytometer between or after being processed, diluted, separated, centrifuged, agglutinated or otherwise altered.

For example, a blood sample or a portion thereof may be prepared for sedimentation or centrifugation for cytometric analysis. The sedimented or sedimented portion of the sample may be resuspended (e.g., by aspiration, agitation, sonication, or other processing method) in a selected buffer prior to performing the cytometric analysis. A biological sample may be diluted or resuspended in water or in a physiological saline solution, such as a buffered saline solution, prior to cytometric analysis. A solution for such dilution or resuspension may or may not include a fixative (e.g., formaldehyde, paraformaldehyde, or other agents that crosslink proteins). A solution used for such dilution or resuspension may provide an osmotic gradient within the surrounding solution and the cells or inner portions of the sample, causing the cellular capacity of some or all of the cells in the sample to change. For example, if the final solution concentration after dilution is lower than the effective concentration in the interior or inner part of the cell, the volume of such cells will increase (e.g., cells will develop edema). A biological sample may be diluted with a solution that may or may not contain an osmotic agent (such as glucose, sucrose, or other sugars; sodium, potassium, ammonium, or other salts; or other osmotically active components or constituents). In an example, an osmotic agent may be effective to maintain the integrity of cells within the sample by, for example, stabilizing or reducing the potential osmotic gradient between the surrounding solution and the interior or interior portions of such cells. In an example, an osmotic agent may be effective to provide or increase an osmotic gradient between a surrounding solution and an interior or inside portion of such a cell effective to at least partially collapse the cell (the interior or inside portion of the cell being less concentrated than the surrounding solution); or effectively edema the cells (the inner or inner part of the cell is more concentrated than the surrounding solution).

For example, a biological sample may be measured or analyzed after a portion of the sample is diluted with a solution containing a dye. For example, a biological sample may be measured or analyzed after a portion of the sample is diluted with a solution containing an antibody or antibody fragment. For example, a biological sample may be measured or analyzed after a portion of the sample is diluted with a solution containing particles. Particles added to a biological sample may be used as a standard (e.g., may be a size standard, the particle size or particle size distribution of which is known; or a concentration standard, the quantity, volume, or concentration of which is known); or may be used as a marker (e.g., may bind or adhere to a particular cell or cell type, a particular cellular marker or cellular component, or bind to all cells in a sample).

For example, a biological sample may be measured or analyzed after one or more types of cells are separated from other cell types. This separation may be accomplished by gravity (e.g., sedimentation), centrifugation, filtration, contact with a substrate (e.g., a surface such as a sidewall or bead containing antibodies, lectins, or other components that may bind or adhere more readily to a type of cell), or other methods. Isolation may be aided or accomplished by changes in one or more cell types. For example, a solution may be added to a biological sample, such as a blood sample, resulting in edema of some or all of the cells in the sample. Whereas edema of one type of cell occurs faster than that of another type or types of cells, it is possible to distinguish cell types by observation and measurement of the sample after addition of the solution. Such observations and measurements may be made at the same time point, or at multiple time points, with the differences (e.g., size, volume, internal concentration, or other characteristics affected by such edema) being selected for emphasis, and with increased sensitivity and accuracy of such observations and measurements. In some cases, one or several types of cells may rupture due to this edema, allowing further observation and measurement of the remaining cell types in the sample.

The observation, measurement, and analysis of a biological sample by a cytometer may include, for example, photoelectric measurements made using a photodiode, a photomultiplier tube, a laser diode, a spectrophotometer, a charge-coupled device, a camera, a microscope, or other methods or devices. Cytometric analysis may include preparing and analyzing (e.g., two-dimensional images) of cells within a biological sample, wherein the cells are labeled (e.g., with fluorescent, chemiluminescent, enzymatic, or other substances), placed (e.g., allowed to deposit on a substrate), and imaged by a camera. The camera may include a lens and may be attached to or used in conjunction with a microscope. Cells may be identified on the two-dimensional image by a label (e.g., a light wave released by a label) attached thereto.

A single cell image, prepared and analyzed by a cytometer, as shown herein may include zero cells, one cell, or multiple cells. Cells within a cytometer image, as shown here, may be labeled, as shown above. Cells within a cytometer image, as shown here, may be labeled, as described above, to effectively identify the image and the material from which the sample was taken.

In some examples, the detection system is designed to perform a cytometric analysis. Cytometric examination is typically used to measure properties of individual cells optically, electrically, or acoustically. For the purposes of the present invention, "cells" may surround non-cellular samples that are generally comparable in size to a single cell, including but not limited to vesicles (e.g., liposomes), small groups of cells, virosomes, bacteria, protozoa, crystals, lipid and/or protein aggregates, and small particle-bound substances such as beads or microspheres. These properties include, but are not limited to, size, shape, particle size, light scattering pattern (or optical index), integrity of the cell membrane, concentration, morphology, and spatiotemporal distribution of cellular contents, including, but not limited to, protein content, protein modifications, nucleic acid content, nucleic acid modifications, organelle content, nuclear structure, nuclear content, intracellular structure, vesicle content (including pH), ion concentration, and expression of other small molecules such as hormones or drugs, markers on the cell surface (including cell membranes and cell walls) including proteins, fats, carbohydrates, and modifications thereof. By using the correct dyes, colorants, or other labeling molecules, which may be in pure form, conjugated or immobilized to other molecules, or bound to nano-or micro-particles, a cytometer may be used to determine the expression, quantitative analysis, and/or modification of a given protein, nucleic acid, lipid, carbohydrate, or other molecule. Properties that may be measured by a cytometer also include measurements of cell function or activity, including but not limited to phagocytosis, antigen expression, cytokine secretion, alterations in expression of internal molecules and surface molecules, binding to other molecules, cells, or substrates, active transport of small molecules, mitosis or meiosis, protein translation, gene transcription, DNA replication, DNA repair, protein secretion, apoptosis, chemotaxis, mobility, adhesion, antioxidant activity, RNAi, protein or nucleic acid degradation, drug response, infectivity, and activity of specific pathways or enzymes. The cytometer may also be used to determine information about a cell population, including but not limited to cell count, total population percentage, and variation in all of the above-mentioned characteristics in a sample population. The assays described herein may be used to measure one or more of these properties in each cell, and it may be advantageous to determine the prototypes or other relationships between different properties. The assays described herein may also be used to measure multiple cell populations independently, e.g., by labeling one moderating cell population with specific antibodies to different cell lines. A microscope module may allow the device to perform histological, pathological, and/or morphological analyses, and may also evaluate objects based on physical and chemical characteristics. The tissue may be homogenized, washed, precipitated onto a cuvette or a slice, air dried, stained (such as with an antibody), incubated, and then imaged. When used in conjunction with the data transfer techniques mentioned elsewhere herein, these inventions facilitate the transfer of images from a CMOS/CDD or similar device to a medical practitioner for review, for example, which is not possible with conventional flow cytometry only devices. The cell counter can measure cell morphology and surface antigen, and can detect the surface antigen more sensitively and specifically compared with the traditional blood laboratory equipment. Interpretation of the cell detection may be automated by gating one or more measurements; such gating thresholds may be set by an expert or learned based on statistical methods from training data; gating strategies may be specific to a single subject and/or study population.

In some instances, the incorporation of a cell counting module into a point-of-care device may provide measurements of cellular attributes, typically measured by general laboratory equipment or laboratories, and the results interpreted by classically trained medical personnel, to improve the speed and/or quality of clinical decisions. Thus, a point-of-service device may be designed for cytometric analysis.

Demonstration 1

A blood sample containing leukocytes including natural killer cells and neutrophils is obtained. The sample is treated with a fluorescently labeled cell identity conjugate (anti-CD 16 conjugate) that binds both natural killer cells and neutrophils. The sample is also simultaneously treated with a nuclear dye (DRAQ 5). The sample was imaged by fluorescence microscopy and dark field microscopy. The fluorescence levels and lateral light scatter levels of different cells within the sample are recorded and analyzed. Segmented images containing anti-CD 16 binder signals provide quantitative information on the fluorescence density (consistent with CD16 expression levels) and cell size per cell. Dark field images provide quantitative information on the scattering properties of each cell. The image containing the DNA staining signal is divided into several parts to determine the fluorescence density, size and shape of the cell nuclei.

As shown in fig. 1A, two main groups of cells were identified based on the measurement of CD16 fluorescent and scattered light signals of different cells. The cell group containing bright/high CD16 fluorescence signal and high scatter signal (fig. 1A, right circle) was neutrophils. The cell group containing the medium CD16 fluorescence signal and the low scatter signal (fig. 1A, left circle) was natural killer cells. Although measuring the fluorescence and scattered light signals of different cells may provide sufficient information to distinguish a large proportion of the cells in a sample as natural killer cells or neutrophils, for some cells, measuring these properties may not provide sufficient information to distinguish these cells with high accuracy. For example, measuring the fluorescent and scattered light signals of cells does not provide enough information to accurately distinguish a small group of cells delineated by small circles (e.g., the middle circle) in FIG. 1A. To identify whether the cells within the small circle are natural killer cells or neutrophils, nuclear staining (DRAQ5) and whole cell (anti-CD 16) staining images were examined. Quantitative measurements of nuclear area and total cell volume are obtained and the ratio of nuclear area to total cell area is determined. As shown in FIG. 1B, the ratio of the nuclear area to the total cellular area of Natural Killer (NK) cells and neutrophils (Neu) was significantly different. Thus, the use of microscopic quantitative analysis techniques to examine various properties of cells within a sample allows us to clearly classify the cells. FIG. 1C shows images of natural killer cells from the smallest circle of FIG. 1A. All images have the same length scale. The left panel is cells stained for the entire cellular area (anti-CD 16), while the right panel is the same cells stained only for the nucleus (DRAQ 5). The top and bottom images are different demonstrations of natural killer cells. FIG. 1D shows an image of neutrophils derived from the smallest circle of FIG. 1A. All images have the same length scale. The left panel is a cell stained for the entire cell area, while the right panel is the same cell stained only for the nucleus. The top and bottom images are different demonstrations of natural killer cells.

In addition, the nucleus of a neutrophil has a pronounced multilobal morphology, while the nucleus of a natural killer cell (and other lymphocytes) is round and smooth. Image segmentation rules may be used to identify and classify cells based on the shape of the cell nucleus itself.

Demonstration 2

A sample containing platelets is obtained. The platelets were labeled with fluorescent conjugated anti-CD 41 and anti-CD 16 antibodies. And a ball of 3 μm diameter was added to the sample. The samples were imaged at 10x and 20x magnification. The fluorescence distribution density of individual platelets (from both antibodies) was measured and confirmed to have a gaussian distribution morphology (fig. 2B). Fluorescence measurements of individual platelets were plotted and a fitted density distribution was determined (fig. 2C). In fig. 2C, the gray line is the measured fluorescence density of each platelet, and the black line is the fitted line. Fitting parameters such as mean gaussian, variance, volume, width, and basal area, etc. can be evaluated as platelet volume predictors. The gaussian volume and fitted width are considered to be most correlated with the mean platelet volume.

In the above measurements, the 3 μm ball functions as a reference and fiducial point to control the variation that occurs when the best focus plane is accurately determined, and the effect of that variation on the volume measurement.

In addition, the estimated platelet size from the fitted two-dimensional model can be calibrated to be within the normal range (fig. 3).

Demonstration 3

A blood sample containing Red Blood Cells (RBCs) is obtained. The RBCs are treated with a low concentration of surfactant (DDAPS or SDS), causing the RBCs to assume a spherical shape. The RBCs were imaged by dark field microscopy in two different cuvettes. (A) One cuvette only allowed for pure epi-illumination (fig. 4A); (B) one cuvette allowed mixed illumination of both epi-and transmission (fig. 4B). RBCs within the mixed illumination cuvette that allow for epi-and transmission are more visible than RBCs within the illumination cuvette that allow for only epi-illumination alone (fig. 4).

Demonstration 4

A sample containing neutrophils is obtained. In neutrophils, the shape and chromosomal morphology of their nuclei may indicate whether the cell is an immature "zonal nucleus" neutrophil or a mature "lobular nucleus" neutrophil. Zonal nuclear neutrophils are immature neutrophils that have just been released from the bone marrow. An increase in the proportion of zonal nuclear neutrophils may indicate a progressive infection or inflammatory state.

The sample is mixed with a fluorescently labeled anti-CD 16 antibody and a cell surface receptor CD16 on neutrophils can be identified. The sample is also stained with a fluorescent nuclear dye. The samples were imaged by fluorescence microscopy, while nuclear staining and CD16 staining data were obtained from the cells. Zonal nuclear neutrophils generally have similar CD16 expression levels as mature lobular nuclear neutrophils, and thus cannot be distinguished from CD16 alone in terms of their fluorescence intensity values.

Image analysis, including segmentation imaging, was used to identify nuclear staining and morphology of zonal and lobular neutrophiles, thus allowing us to classify neutrophils. The size, morphology and fluorescence intensity of the nuclei were measured. Furthermore, the nucleus is analyzed to determine the number of leaves (peak density of the nucleus region), the distance between two leaves of the nucleus, and the change in the contour curve (second derivative) of the nucleus. FIG. 5A shows a representative banded nuclear neutrophil image. In these images, the nuclei appear in a light gray while the cytoplasm appears in a dark gray. Since neutrophils are differentiated from myeloid stem cells, they develop a characteristic "U" shaped nucleus before they reach full maturation. FIG. 5B shows a representative lobulated nuclear neutrophil image. In these images, the nuclei appear in a light gray while the cytoplasm appears in a dark gray. The nucleus of a segmented nuclear neutrophil contains multiple segments per segment (typically 3-5). Thus, this analysis facilitates the identification and quantification of different neutrophil subpopulations in blood.

Demonstration 5

A sample of cells was obtained from a patient with Chronic Lymphocytic Leukemia (CLL). The goal of the study was to quantify the expression of CD5 in B cells of patients. anti-CD 20 antibody was selected as a B cell binder. An anti-CD 20 antibody that is fluorescently labeled with a first color is mixed with the sample. After a suitable incubation period, the sample is washed and unbound anti-CD 20 antibody is removed. The sample is exposed to a light source that activates the first fluorescence and the fluorescence signal is measured using a spectrophotometer. Based on the measured fluorescence signal, an approximate concentration of B cells in the sample can be determined. The approximate concentration of B cells determined is in fact 1.5 times the true concentration of B cells in the sample.

The correct amount of an anti-CD 5 conjugate was added to the sample based on the approximate concentration of B cells in the sample to maintain a proper ratio of CD5 expression to CD5 fluorescence. The anti-CD 5 conjugate was coupled to a second fluorophore having a different peak activation wavelength than the first fluorophore attached to the anti-CD 20 conjugate. The anti-CD 5 antibody is added to the sample, and the individual cells in the sample are then exposed to a light source that activates a second fluorescence, and the fluorescence signal of the individual cells is measured. From the fluorescence signal measured from the cells, the average amount of CD5 in the sample B cells can be determined.

Although the example is described in the context of CD5, it should be understood that obtaining a correct amount to guide the addition of a desired amount of material for use in subsequent steps is not limited to CD5, except that the concept is used with other types of cells, assay substrates, or targets.

Demonstration 6

Blood cells may be imaged, confirmed, and quantified according to the methods described herein. For example, a two-dimensional image of cells in a biological sample, wherein the cells are labeled (e.g., with fluorescent, chemiluminescent, enzymatic, or other substances), placed (e.g., allowed to deposit on a substrate), and imaged by a camera, may be prepared and analyzed as described in the present example. The camera may include a lens and may be attached to or used in conjunction with a microscope. Cells may be identified on the two-dimensional image by a label (e.g., a light wave released by a label) attached thereto.

80 microliters of whole blood obtained from a fingertip blood collection was loaded into a lidded sample container pre-containing 2mg/ml EDTA. In this example, a closed sample container (with a removable or pierceable cap) is used; it should be understood that any suitable container for holding such small volumes of sample may be used, including but not limited to a lidded container or an uncapped container. The sample container was centrifuged at 1200x g for 5 minutes to separate the blood cells from the plasma. Centrifugation of the sample container causes the blood sample within the sample container to separate into two major portions (from the top to the bottom of the sample container). 1) Plasma, and 2) packed blood cells. This procedure ensures that no drop of blood remains in its liquid portion after separation. Moreover, this process separates the cells from the plasma component, which can reduce metabolism and allow for longer storage of the sample.

The centrifuged sample container is placed in a cassette containing a plurality of liquid separation reagents, a tip and a cell counting cuvette. The cassette contains all the reagents required for detection. The cartridge is loaded into an apparatus equipped with at least a centrifuge, a pipette and a platform to load the cuvette. The pipette in this device has a plurality of nozzles, some of which are of different sizes than others.

In this apparatus, a nozzle on the pipette is lowered onto a cuvette loading tool to engage an associated hole in the cuvette loading tool. The tool is then moved to the cassette and lowered onto a cell counting cuvette. The pin on the tool can engage and lift the corresponding hole on the cuvette. The cuvette is transferred to a loading platform located elsewhere in the apparatus.

Next, a larger nozzle of the pipette is lowered inside the device into the cassette, engaging a pipette tip stored in the cassette. The pipette is used together with a tip to mix cells and plasma in a sample container, and the pipette tip is placed in the sample container to repeatedly suck in and discard the material from the tip. Once the cells were resuspended in plasma, the whole blood sample was thoroughly mixed and 5 microliters of mixed whole blood was aspirated to provide a aliquot sample for measurement of the characteristics of the blood sample. These 5 microliter aliquots were used for measurements of red blood cells and platelets in the samples. The remaining portion of the 5 microliter aliquot removed was used for the measurement of leukocytes in the sample, as described below.

Whole blood was diluted 20-fold by placing 5 microliters of whole blood in a container containing a mixture of phosphate buffered saline and 2% bovine serum albumin (resulting in a 100 microliter diluted sample). After vigorous mixing, 5 μ l of the sample was transferred to another mixture containing the following labeled antibodies: alexa647(AF647) conjugated anti-CD 235a, Phycoerythrin (PE) conjugated anti-CD 41 and anti-CD 61. The mixture was incubated for 5 minutes. Next, 10. mu.l of the mixture was mixed with 90. mu.l of a buffer containing a zwitterionic surfactant to prepare a mixture<0.1% by weight of the components were mixed. The surfactant molecule alters the binding properties of red blood cells in that all cells assume a stable spherical shape. This change is isovolumetric, since the buffer used is isotonic with the cytoplasm; so that no permeability-driven fluid exchange across the cell membrane occurs. After another 2 minutes incubation, 30 microliters of this solution was mixed with a solution containing glutaraldehyde, a fixative and 10um diameter non-fluorescent beads. The final concentration of the mixture was 0.1% glutaraldehyde and 1000 beads per microliter. Glutaraldehyde fixes cells rapidly to prevent cell lysis and other active biological processes.

In this non-limiting example, the pipette engages a tip in the cartridge, aspirates 7 microliters of the mixed solution, and loads the 7 microliters of mixed solution into a tube of the cuvette, which is placed on a platform with the carrier. After loading the mixture into the cuvette, the pipette aspirates 10 microliters of mineral oil from a container in the cartridge and then drops a drop of mineral oil at two openings of the cuvette loading tube. The addition of hexadecane to the open port of the tube prevented evaporation of liquid from the loaded cuvette tube (mineral oil also has this effect). Next, the device-level sample processing device engages the cuvette transporter/cuvette combination and transfers the cuvette transporter/cuvette combination from the cartridge-containing module to the cell counting module of the device. At the cytometry module, the instrument-level sample processing device places the cuvette transporter/cuvette combination on the microscope stage of the cytometry module. The time required for these procedures, plus a 2 minute wait time, allowed the swollen cells to settle to the bottom of the cuvette prior to imaging.

After the cuvette transporter/cuvette combination is placed on the microscope stage, the stage is moved to a predetermined position to allow the optical system of the cytometer to view an opening of the tube containing the sample. In this position, the optical system will relay the sample image obtained by an annular dark field illumination. These images are coupled with the optical system drive on a pivot located perpendicular to the plane of the cuvette for finding the best plane of focus. Once the focusing is successful, the optical system is used to acquire fluorescence images of the sample at different wavelengths, comparable to the fluorophore wavelength used. For example, to see Alexa conjugated by anti-CD 235647 labeled red blood cells, a red light (630nm wavelength) is used to activate the sample and light having a wavelength between 650nm and 700nm is used to image the sample. A polychromatic optic and a bandpass filter combination are used to filter unwanted wavelengths in the optical signal. Since the cells have settled at the bottom of the cuvette, imaging on a single focal plane is sufficient to see all the fine particles in that region And (4) cells.

Data from the images is processed by a controller associated with the sample processing device. The image processing algorithm used here detects cells by their fluorescence image using a combination of adaptive threshold and edge detection. A region of interest (RoI) is created around each cell according to the local density and density gradient. When imaging with dark fields, the beads in the sample were also confirmed and RoIs were created around the beads. All RoIs in each field were counted and the fluorescence density of each image in that field was calculated. The information output by the image processing algorithm includes collision or morphological measurements and fluorescence and dark field densities for each RoI. This information was analyzed statistically to classify each target as a red blood cell (CD235a positive, but CD41/CD61 negative), a platelet (CD41/CD61 positive and CD235a negative), and a bead. These shape description indices, such as perimeter, diameter, and circularity, are used to calculate the volume of each red blood cell and platelet. Since the beads are added to the sample at a known concentration, the average ratio of beads to cells throughout the tube is used to calculate the cell concentration in cells/microliter. This concentration is dilution corrected to obtain the concentration of cells in the original whole blood sample, according to the sample processing step. The following is a quantitative analytical calculation for one sample: 1) the number of red blood cells in the cuvette; 2) the average volume of red blood cells in the cuvette; 3) the width distribution (RDW) of the red blood cells in the cuvette; 4) the number of platelets in the cuvette; and 5) the average platelet volume in the cuvette. The following are calculations performed on the original blood sample based on these calculations:

Measured value	Results	Typical range
			Concentration of erythrocytes (million/microliter)	4.8	4-6
Mean volume of erythrocytes (Fei Sheng)	88	80-100
			Erythrocyte Width distribution (RDW) (%)	12	11-14.6
Platelet concentration (thousands/microliter)	254	150-400
			Platelet mean volume (Fei Sheng)	10.4	7.5-11.5

After removing 5 microliters of the aliquot sample for RBC and platelet information analysis, the remaining 75 microliters of sample was used for analysis of leukocytes. The remaining 75 microliters of whole blood was also mixed with the sample by repeated pipetting and releasing in the same container using a pipette. Approximately 40 microliters of the remaining mixed 75 microliters of whole blood sample is pipetted into a pipette tip and transferred by the pipette into a centrifuge tube of the cartridge. The tube containing the blood sample is transferred and placed in a float bowl in a centrifuge in the module after engaging the pipette. The centrifuge was spun at 1200x g for 3 minutes to separate the blood sample into EDTA-containing plasma supernatant and packed cell pellet.

After centrifugation, the tube is removed from the centrifuge and returned to the cassette. The plasma supernatant is removed by the pipette and transferred to a different reaction vessel in the cartridge. From a reaction vessel in the cartridge, 16 microliters of resuspension buffer was aspirated by the pipette and added to the cell pellet in the centrifuge tube. The pipette resuspends the cell pellet in the resuspension buffer by repeatedly aspirating and releasing the mixture from the centrifuge tube. Next, the pipette aspirates 21 microliters of resuspended whole blood and adds it to another pipette containing 2 microliters of Pacific Blue ^TMAndconjugated anti-CD 14 in containers, mixed and incubated for 2 minutes. 20 microliters of the mixture was added to an 80 microliter lysis buffer. The lysis buffer is a mild surfactant solution, such as a saponin, used in conjunction with a fixative, such as a paraformaldehyde. The detergent causes a large number of pores in the cell membrane. Red blood cells, due to their unique membrane properties, are particularly sensitive to such pore formation and are completely lysed, with the contents leaking into the surrounding liquid. Fixatives are added to prevent unnecessary lysis of the leukocytes. Platelets also remain unlysed. The purpose of this step is to remove red blood cells from the mixture, since they far exceed the number of white blood cells in a ratio of about 1000: 1. Platelets have no effect on the imaging and are therefore not relevant to this process. The lysis buffer also contained a known concentration of 10. mu.M non-fluorescent beads.

After 5 minutes of incubation, the vessel was again centrifuged at 1200x g for 3 minutes. The supernatant was aspirated by a pipette tip, red blood cells and other cellular debris were removed, and placed in a waste area within the cassette. In the pelleted cells, the packed white blood cells were in approximately 15 microliters of liquid.

To determine the approximate number of leukocytes in the deposited cells, the leukocytes are first resuspended in the container using the pipetteThe liquid is then aspirated and transferred to a spectrophotometer for detection. The leukocyte suspension is illuminated with light of 632nm wavelength, Alexa647 of dyes andthe activation wavelength. The light waves released by the cell suspension were filtered through a 650nm long pass filter and measured in the spectrophotometer. This measurement corresponds to a previously generated calibration curve to estimate an approximate concentration of leukocytes in the cell suspension. Typically, the cell concentration is in the range of about 1000 cells/ml to 100000 cells/ml. This estimate is used to calculate a correct dilution factor to ensure that the cell concentration in the cuvette is limited to within twice the preset target density. The purpose of this step is to ensure that the density of cells contained in the cuvette is not too high or too low. If the cell density is too high, the accuracy of image processing operation is impaired; when the density is too low, the number of cells in the sample is insufficient.

A labeled antibody containing anti-CD 45 (leukocyte marker), CD16 (neutrophil marker), and CD123 (basic granulocyte marker) was added to the cell suspension and mixed according to the dilution factor calculated in the step.

Once the cuvette complexed with the cuvette carrier was placed on the cuvette carrier plate, 10 μ l of the leukocyte mixture resuspended in cell counting buffer was loaded into both tubes in the cuvette. After loading the mixture into the cuvette channel, the pipette pipettes 10 microliters of hexadecane from one container in the cartridge and then drops a drop of mineral oil at both openings of the cuvette channel for loading white blood cells.

Next, the device-level sample processing device engages the cuvette transporter/cuvette combination and transfers the cuvette transporter/cuvette combination from the cartridge-containing module to the cell counting module of the device. At the cytometry module, the instrument-level sample processing device places the cuvette transporter/cuvette combination on the microscope stage of the cytometry module. After placing the cuvette transporter/cuvette combination on the microscope stage, the two tubes of the cuvette containing the leukocytes were imaged, as described for RBC/platelets.

Dark field imaging of leukocytes was used to count the number of leukocytes in one field (as shown in fig. 9A). Cell surface markers are used to determine the cell type of individual leukocytes in an image; for example, CD14 labels monocytes; CD123 labeling basophils; CD 16-labeled neutrophils; whereas CD45-AF647 was used to label all white blood cells (fig. 9B-9E) the nuclear stain Draq5 was used to label the nucleus and distinguish nucleated cells (such as white blood cells) from mature red blood cells, which have no nucleus (fig. 9F).

The image processing algorithm used here detects cells by their fluorescence image using a combination of adaptive threshold and edge detection. A region of interest (RoI) boundary is created around each cell according to the local density and density gradient. Using dark field imaging, the beads in the sample were also confirmed and a RoIs limit was created around the beads. All RoIs in each field were counted and the fluorescence density of each image in that field was calculated. The information output by the image processing algorithm includes collision or morphological measurements and fluorescence and dark field densities for each RoI. This information is analyzed by statistical methods to classify each target as a lymphocyte, monocyte, basophil, eosinophil, neutrophil, or a rolling ball. Based on the number of different types of cells, the number of beads and the dilution ratio corresponding thereto during sample processing are implemented and the absolute concentration of one cell per microliter of the original whole blood is calculated. This method was used for all leukocyte and each subtype calculation and reported in absolute concentration (number of cells per microliter) and percentage (%).

An example of an image and graph for each measurement is shown in fig. 9, 10 and 11.

FIG. 9 shows a representative blood cell image from a whole blood sample; these images were taken by different imaging techniques and staining. The image shown in fig. 9A is a cell in a whole blood sample taken using dark field illumination. FIG. 9B shows an image taken of whole blood sample cells showing fluorescent visualization of Pacific Blue dye-labeled anti-CD 14 antibody; the fluorescent cells are monocytes. FIG. 9C is a photograph taken of whole blood sample cells showing fluorescent visualization of PECy5 dye-labeled anti-CD 123 antibody; the fluorescent cells are basophils. FIG. 9D is a photograph taken of whole blood sample cells showing fluorescent visualization of PE dye-labeled anti-CD 16 antibody; the fluorescent cells are neutrophils. FIG. 9E shows an image taken of whole blood sample cells showing fluorescent visualization of AF647 dye-labeled anti-CD 45 antibody; all leukocytes were under this fluorescent image. The image shown in FIG. 9F was taken of a whole blood sample cell coverThe nuclei were stained. Thus, white blood cells and platelets are stained and show fluorescence, while red blood cells (lacking nuclei) are not stained and show no fluorescence.

FIG. 10 shows a representative blended image of cell types in whole blood cells, the image obtained according to the methods shown herein. Shown are a monocyte image (seen in the upper left of the figure, labeled as a pale red surrounded by a blue-violet ring outside the center), a lymphocyte image (seen in the center of the figure, labeled as a bright red surrounded by a dark red ring outside the center), an eosinophil image (seen in the lower left of the figure, labeled as a green surrounded by a red border), and a neutrophil image (seen in the lower right of the figure, labeled as a green surrounded by a yellow-green border).

It is of great interest to identify and quantify the types of cells found in similar blood samples. There may be multiple ways to perform a similar classification process, and in some instances, such multiple classifications may be considered a statistical problem. It will be appreciated that there are a number of methods provided in the art for addressing these cell sorting problems. One particular example of such an analysis is provided below.

FIG. 11 shows a graph of different cell types identified and quantified by the cytometric assay described in this example. FIG. 11A shows a spot pattern (cells) of FL-9 (dark field scattered signal) density vs. FL-17 (pacifiic blue dye labeled anti-CD 14 antibody) density for the identification of monocytes. FIG. 11B shows a dot pattern (cells) of marker FL-19(PE-CY5 dye-labeled anti-CD 123 antibody) versus marker FL-15(PE dye-labeled anti-CD 16 antibody) for basophil identification. FIG. 11C shows a spot pattern (cells) displayed by marker FL-15(PE dye-labeled anti-CD 16 antibody) density versus marker FL-11(AF647 dye-labeled anti-CD 45 antibody) density for lymphocyte validation. FIG. 11D shows a spot pattern (cells) of FL-9 (dark field scattered signal) density vs. FL-15(PE dye labeled anti-CD 16 antibody) density for identification of neutrophils and eosinophils.

Initial confirmation of monocytes (9.6% as shown in fig. 11A) was used to guide subsequent basophil confirmation (0.68% as shown in fig. 11B). The confirmation of monocytes and basophils, as shown in FIGS. 11A and 11B, was used to guide the subsequent confirmation of neutrophils and eosinophils (68% neutrophils and 3.2% eosinophils in WBCs, as shown in FIG. 11D). Finally, confirmation of lymphocytes is shown in fig. 11C (93% of WBCs are shown in fig. 11C, corresponding to 18% of cells in the original sample).

The current method correlates well with other methods. White blood cells, red blood cells, and platelets were counted using EDTA anticoagulated whole blood samples. The white blood cells are further counted to determine the number of neutrophils, monocytes and lymphocytes in the sample. In the measurement method shown in fig. 12, a sample of EDTA-anticoagulated whole blood was divided into 2 portions, a portion of which was run in the system of the present invention and analyzed using the method shown here. While another portion of the sample was run in the Abbott CELL-DYN Ruby system (Abbott Diagnostics, Lake Forest, il, usa) and analyzed by a commercial multi-parameter automatic blood analyzer. A comparison of the results obtained with the two methods is shown in figure 12.

As shown in fig. 12A-12C, the numbers of white blood cells ("WBCs", fig. 12A), red blood cells ("RBCs", fig. 12B), and platelets (fig. 12C) measured by the current method correlated very well with WBCs, RBCs, and platelet number measurements of the same corresponding aliquot of sample analyzed by the current method by other methods. As shown in fig. 12D-12F, the numbers of neutrophils, monocytes, and lymphocytes measured by the two methods are very close and correlated with each other very well.

As used herein, the term "cell count" refers to the observation, analysis, identification, and results of cells in a biological sample, either substantially in a liquid or on a substrate. Cells detected and analyzed by the cytometer may be detected and measured by any optical, electronic, or acoustic detector. A cytometer may include preparing and analyzing an image (e.g., a two-dimensional image) of cells within or derived from a biological sample. The cells may be labeled (e.g., with fluorescent, chemiluminescent, enzymatic, or other labels) and plated (e.g., to allow deposition on a substrate) and are typically imaged by a camera. A microscope may be used to image cells in a cytometer, for example, cells may be imaged by a camera and a microscope, for example, an image of a microscope image taken by a camera. An image formed by or for a cytometer typically includes more than one cell.

Optical system

Referring now to fig. 6A and 6B, an example of an optical system suitable for use herein will now be described. While these examples of the system are described at a cytometer, it should be understood that these examples of the system may also be used in more than a cytometer. By way of non-limiting example, the imaging and imaging processing capabilities of the system shown herein may have a variety of applications, including applications other than cytometers. Since the sample image for analysis is taken and the image information is typically linked to or associated with the system for quantitative measurements, we can further analyze the quantitative information-related images to collect unreported clinical information in the image.

A sample analyzed by, for example, a cytometer or other optical or imaging methods, may be retained in a sample holder for analysis. For example, a cuvette may be used as such a sample holder. The example of fig. 6A shows a perspective view of a cuvette 600 having a plurality of openings 602 for receiving a sample or portion of a sample for analysis. The horizontal cross-sectional shape in the example of fig. 6A resembles a rectangle. Although the system is described in the context of a cuvette, it should be understood that other sample receiving devices may be used in place of the cuvette 600, or in conjunction with the cuvette 600.

As seen in the example of fig. 6A, the openings 602 may allow a sample containment system (not shown) or other transport system to place samples within the openings 602, which may be connected to or lead to an analysis region 608 within the cuvette where the sample may be analyzed. In one non-limiting example, an analysis region 608 may be a chamber. In another non-limiting example, an analysis region 608 may be a pipe. In an example, one analysis region 608 designed as a conduit may be in communication with both inlets 602. In a further non-limiting example, an analysis region 608 may be a conduit that retains the sample in a non-flowing manner. In any of the examples herein, the system can retain the sample in a non-flowing manner during analysis. Alternatively, some other examples may be designed to allow the sample to flow through the analysis region before, during, or after analysis. In some examples, after analysis, the sample is aspirated from the cuvette 600 and then transferred to another platform (in a system containing multiple platforms) for further processing and/or analysis. Some examples may use a gate in the system to control the flow of the sample.

Fig. 6A shows that in some examples a cuvette 600 may contain a plurality of openings 602. Samples may be added to the sample holder through inlet 602. One opening 602 may be operatively connected (e.g., in fluid continuity) with one analysis region 608. One analysis region 608 may be operatively connected (e.g., in fluid continuity) with a plurality of openings 602. It should be understood that some examples may include more, or fewer, openings 602 in the cuvette 600 some examples may be associated with a particular opening 602 such that a selected mating or mating opening 602 may enter the same channel (e.g., an analysis region 608 designed as a channel). By way of non-limiting example, there may be one opening 602 at each port of one analysis region 608. Alternatively, there may be more than one opening 602 at a port of one analysis region 608.

A cuvette 600 in one example may contain structures 610 that allow a sample processing system to engage and transport the cuvette 600. A cuvette 600 shown in fig. 6A and 6B may interface with a sample processing system via an element 610, with the effect that the cuvette 600 may be transported from one location to another. An element 610 may also be used to ensure that a cuvette 600 is in a desired position, for example, before or after being transported to a location (e.g., a detector for optical imaging and analysis), a cuvette 600 may be held in a position by an element 610, or a cuvette 600 may be held in a position by a tool or device that uses an element 610. In one non-limiting example, the structure 610 can be an opening in the cuvette 600 that allows a pipette or other elongated member to engage the cuvette 600 and transport it to a desired location. Alternatively, the structure 610 may be, or may include, a protrusion, a hook, a magnet, a magnetized element, a metallic element, and/or other structures that may be used to engage a cuvette transport apparatus in place of or in combination with the opening. In one example, external forces (e.g., pressure, or other forces) may act on a cuvette 600; for example, pressure may act on a cuvette 600 to press the cuvette 600 against a floor or surface (e.g., a surface of a substrate support 620) that effectively brings the cuvette 600 into optical contact with the surface. In an example, such external forces (e.g., pressure) may help provide desirable optical properties, such as providing good contact between a cuvette 600 and a substrate support 620, effectively allowing light to pass through or generate other desired optical properties without significant distortion or reflection at the interface. In an example, such an external force (e.g., pressure) may be implemented, at least in part, by one structure 610 or by multiple structures 610.

As shown in fig. 6B (transparent view), a cuvette 600 may have a circular horizontal cross-sectional shape. An opening 602 (or openings 602, which may be present in the same example, not shown) may allow a sample processing system or other transport system to place a sample within the opening 602, which may lead to an analysis region 608 within the cuvette, where the sample may be analyzed. Non-limiting examples of suitable analysis regions 608 include an analysis region 608 comprising a chamber, and an analysis region comprising a conduit. In an example, such an analysis region 608 may be located within a ring structure, such as ring structure 604 shown in fig. 6B. In an example, one opening 602 may be connected to one analysis region 608. In an example, an analysis region 608 within a structure 604 may form a continuous annular chamber and be connected to an opening 608, effectively allowing sample to flow in the chamber from an opening 602 in either direction. In an example, an analysis region 608 within a structure 604 may form an annular channel or chamber, one end of which is connected to an opening 608 and the other end of which is disconnected or sealed from the opening 602, effectively allowing only one-directional flow of sample from the opening 602 within the chamber. In examples, the unidirectional annular conduit or chamber may have a vent or other hole at a location remote from an opening 602. In a further non-limiting example, the analysis zone may be or may include a conduit for retaining the sample therein in a non-flowing manner; a sample may be retained in a non-flowing manner within an analysis region 608 comprising an annular channel that is either bi-directionally connected to an opening 602 or is only uni-directionally connected to an opening 602. In any of the examples herein, the system can retain the sample in a non-flowing manner during analysis. Alternatively, some other force may be designed to allow the sample to flow through the analysis region before, during, or after analysis. In some examples, after analysis, the sample is aspirated from the cuvette 600 and then transferred to another platform (in a system containing multiple platforms) for further processing and/or analysis. Some examples may use a gate in the system to control the flow of the sample.

FIG. 6B shows only a single ring structure 604; however, it should be understood that in the example of a cuvette 600 shape as shown in FIG. 6B, a cuvette 600 may contain a plurality of ring-shaped structures 604. For example, a cuvette 600 containing a plurality of ring structures 604 may contain concentric ring structures 604 of different sizes, with a peripheral ring structure 604 surrounding one or more inner ring structures 604. Such ring structures 604 may include an analysis region 608 within each ring structure 604. FIG. 6B shows only a single opening 602; it should be understood, however, that in a further example of the shape of a cuvette 600 as shown in FIG. 6B, a cuvette 600 may contain a plurality of openings 602. For example, a cuvette 600 comprising a plurality of ring structures 604 (e.g., comprising a plurality of concentric ring structures 604) may comprise a plurality of openings 602 (e.g., each ring structure 604 may comprise at least one opening 602). It should be understood that some examples may include more, or fewer, openings 602 in a cuvette 600. Some instances may be associated with a particular opening 602 such that a selected mating or mating opening 602 may enter the same conduit or chamber. By way of non-limiting example, there may be one opening 602 at each port of an analysis zone. Alternatively, there may be more than one opening 602 at a port of one analysis region 608.

Some examples of cuvettes shown in fig. 6A and 6B may provide structure 604 for selected regions of a cuvette 600. In one example the structure 604 is in the form of raised striations that provide structural support to a region within the cuvette selected to have a controlled thickness (e.g., region 613). For example, the thickness may be selected to provide desired optical properties, including a desired path followed by light before and after reflection within the cuvette 600. Such reflection may be Partial Internal Reflection (PIR) or Total Internal Reflection (TIR). Whether such reflection occurs depends on a number of factors including the wavelength of the light, the angle at which the incident light reaches a surface, the composition of the material (area 613 and the material surrounding an area 613 that activates the peripheral wrap), and other factors. In the example shown in fig. 6A, the structure 604 is rectangular and has a rectangular cross-section. In the example shown in fig. 6B, the structure 604 is annular and may have a rectangular cross-section, or a diamond-shaped cross-section, or other shaped cross-section. These structures may have any suitable cross-sectional shape. As shown in fig. 8B, the structure 604 may have a triangular cross-section (e.g., if it is in the form of a plurality of raised stripes, a saw-tooth cross-section is formed). It should be understood that such structures 604 may comprise other shapes and cross-sectional shapes (e.g., semi-circular, elliptical, irregular, or otherwise), and that in an example, more than one representation of a shape may be present within the same system (e.g., a cuvette may comprise a rectangular, triangular, or otherwise shaped structure). When the controlled thickness region 613 is reduced in thickness relative to a particular region of the cell, the structure 604 may be used, which may benefit from the mechanical support provided by the structure 604.

In addition to providing structural support, the structure 604 may help provide materials and pathways for internal reflection of light within one cuvette 600. As shown in fig. 8A-8D, the light reflection within a cuvette 600 may include a path for light reflection within a structure 604 (e.g., a raised stripe structure or a structure having a triangular cross-section, as shown, or any other shape, such as a circular or semi-circular cross-section, or other cross-sectional shape). Such that the structure 604 may provide a convex structure extending outwardly from a surface 614 of a cuvette 600; or it may be possible to provide concave structures extending inwardly from a surface 614 of a cuvette 600; or it is possible to provide both convex and concave structures from one surface 614 of one cuvette 600. Thus, structure 604 may provide mechanical support for one cuvette 600; may provide desirable optical characteristics for one cuvette 600; it is also possible to provide a cuvette 600 with other desired and useful structures and functions, as shown herein.

The support row structure 604 may thus be used to provide structural support, including stiffness support, for one cuvette 600. The optical properties of a cuvette 600 can be important for its use in optical imaging and other optical measurements of cells, particles or other components in a sample, and similar samples, in an analysis region 608. Maintenance of the flatness of a surface of a cuvette 600, including maintenance of the flatness of a base portion 606, or a surface 614 or 618; maintenance of the proper orientation and configuration of a cuvette 600 (e.g., no twisting, bending, or other twisting); and maintenance of the proper position of a cuvette 600 (e.g., on a base support 620 or within an optical setup) can be important for the integration of optical measurements and imaging using the cuvette 600. Thus, the design and configuration of, for example, the supportive structure 604 and the base portion 606 can be very important factors in providing and maintaining the correct optical properties of a cuvette 600. The maintenance of the correct dimensions of an analysis region 608, including the correct distance and relative angle between the top and bottom surfaces (or sidewalls) of an analysis region 608, can be important to provide correct and consistent illumination of a sample within an analysis region 608. Maintaining the correct dimensions of an analysis region 608 may also be important in ensuring the volume of an analysis region 608, i.e., the volume of sample within the analysis region 608. As discussed herein, external forces (e.g., pressure) may be applied to a cuvette 600 to further ensure proper flatness, or to reduce twisting or twisting, or to ensure proper shape, size, and orientation of a cuvette during use. It will be appreciated that external pressure may not be required to ensure this correct flatness, and thus the correct shape, size and orientation of a cuvette during use. For example, in an example, the individual structures 604 may already be sufficient to help or ensure that a cuvette 600 has the correct flatness, and the correct shape, size, and orientation during use. It should also be understood that in an example, a mere external pressure may have been sufficient to assist or ensure this proper flatness, as well as the proper shape, size, and orientation of a cuvette during use. It should be appreciated that in an example, the combination of the structure 604 and the external pressure may help or ensure proper flatness and maintenance of a proper shape, size, and orientation of a cuvette during use.

A cuvette 600 including a supporting structure 606 and a cover portion 612 may be made of any material having suitable optical properties. In an example, a cuvette 600 including a support structure 606 and cover portion 612 may be made of glass (e.g., quartz, or borosilicate glass, or aluminosilicate glass, or sodium silicate glass, or other glass). In examples, a cover portion 612 or a substrate support portion 620 may be made of an acrylic plastic, or a colorless polymer (e.g., a cyclic olefin copolymer, a polycarbonate, a polystyrene, a polyethylene, a polyurethane, a polyvinyl chloride, or other polymer or composite polymer), or other transparent material. In addition to the optical properties of this material, its physical properties (e.g., hardness, strength, melting point, likelihood of being machined, and other properties), compatibility with other materials, price, and other factors may also affect the choice of materials used to make a cuvette 600. As discussed above, the manifestation of structure 604, the availability of extraneous pressure (e.g., that may be imparted by one structure 610 or applied directly to at least a portion of structure 606 and cover portion 612), and other factors may allow the use of materials, such as materials having a lower hardness than quartz, that may also provide the optical and mechanical properties required in the systems and methods illustrated herein. In addition, the manifestation of structure 604, the availability of external pressure, and other factors may allow for the use of some manufacturing and durability techniques that may be impractical in the absence of similar structures, pressure, and other structures (e.g., due to deformation or other factors). In addition to the manifestation of structure 604, the availability of external pressure, and other factors may allow for the use of materials, including inexpensive materials, that may not be possible in the absence of similar structures, pressure, and other structures.

Therefore, proper design, construction, and use of materials to support the row structures 64 and base portion 606 is very important to the cuvette 606 and its use.

In some examples, the thickness control regions 613 (see figures 8A, 8B, and 8D) are selected to be placed above the analysis region 608. In some examples, the thickness control regions 613 can impart particular optical properties to or near the analysis region. Some examples are also possible to design the structure 604 to provide some optical properties to the light passing through the cuvette 600. Alternatively, in some instances, the structure 604 may be designed to not have any effect on the optical quality of the cuvette 600. In such an example, the structure 604 may be designed to contain one or more optically absorbing surfaces. For example, without limitation, such a particular surface may be black. Alternatively, some examples may have the structure 604 formed of a light absorbing material. Alternatively, the structure 604 may be placed in a position to provide mechanical support to the cuvette 600 near the analysis region, but not to interfere with its optical properties.

For example, certain surfaces, including a surface 614 of a thickness control region 613 and a surface 618 of a structure 604, may be coated with a black or other color. Such a coating may comprise one coating layer or may comprise a plurality of coating layers. For example, coatings suitable for one surface 614 or 618 may include 2, 3, 4, 5, 6, 7, or more coatings. In examples, a surface (such as a surface 618) or a surface 614 of a structure 604 may be covered with 3 or 5 coatings, for example. Such a coating may include a dye, an ink, a paint, a surface treatment, a colored tape, or other coating or surface treatment techniques. In the example, a black or other colored logo (e.g., a Paper) OrOr MagicOr other indicia) may be used to coat a surface 614 of a thickness control region 613, or a surface 618 of a structure 604. For example, an oversized black marker may be used to apply a multi-layer coating of black ink to a surface 614 or an outer surface 618 of a structure 604 to provide an optically absorbing surface to improve the optical quality of a cuvette 600. In an example, one surface 614 or 618 may be coated or treated to affect or reduce the reflection (PIR or TIR) of that surface. A reduction in reflection from a surface may affect (e.g., reduce) background illumination from a surface.

In examples, certain surfaces, including a surface 614 of a thickness control region 613, and a surface 618 of a structure 604, may be coated or covered with a material that enhances the reflectivity of the surface. By, for example, coating a surface or attaching a material to a surface, the reflection of a surface may be enhanced; suitable materials for enhancing reflection include aluminum, silver, gold, and dielectric materials (e.g., magnesium fluoride, calcium fluoride, or other oxide salts or metals, or other reflective or dielectric materials). Such a coating or covering may comprise one coating layer or may comprise a plurality of coating layers. For example, coatings and coverings suitable for one surface 614 or 618 may include 2, 3, 4, 5, 6, 7, or more coatings. An increase in reflection from one surface may affect (e.g., increase) transmitted illumination from one surface. An increase in surface reflection may aid or enhance imaging of a sample within an analysis region 608; or may assist or enhance the optical analysis of a sample within an analysis region 608.

It should be understood that the cuvette 600 is typically made of an optically transparent or transmissive material. Alternatively, only selected portions of the cuvette 600 (e.g., the analysis region or a region associated with the analysis region) may be made of an optically transparent or transmissive material. Alternatively, selected layers or regions of the cuvette 600 may be designed to be non-light transmissive. A portion or region of a cuvette may be covered or coated to absorb light; for example, a surface (or portion) may be coated with a dark color, or a light absorbing dye or ink. In a further example, a surface (or a portion) may be coated with a dark color, or an absorbing coating, such as a dark or absorbing material, e.g., tape, or cloth, or paper, or rubber, or plastic.

6A, 6B, and 8A-8D show examples in which the cuvette 600 is placed on a base support 620, and a portion or all of the base support 620 is made of an optically transparent or transmissive material. In some examples, such optically transparent or transmissive material portions are designed to be in line with the analysis region of the cuvette 600 to allow optical data acquisition of the sample within the analysis region. In a non-limiting example, the base support 620 can be moved in X, Y, and the Z-axis, to move the cuvette 600 to a desired position for imaging. In some examples, the base support 620 comprises a platform or stage that is movable in only two axes. Alternatively, some support structures may only be movable in a single axial direction. The cuvette 600 may be designed to be operatively coupled to the support structure 600 by friction, mechanical coupling, or by retaining features mounted to one or both of the components. In an example, pressure or other external forces may be applied to one cuvette 600 and/or one base support 620 to ensure proper contact and proper engagement between one cuvette 600 and one base support 620. In examples, such pressure may help ensure that an optically transmissive surface of a cuvette 600, or a base support 620, or both, is optically planar and substantially free of distortion. For example, in an example, a cuvette 600 may be pressed against a base support 620 to reduce or avoid any possible optical distortions caused by imperfections or anomalies in an optical surface of a cuvette 600. In an example, such external forces (e.g., pressure) may help provide desirable optical properties that effectively allow light to pass through that would otherwise distort at the interface surface. In an example, such an external force (e.g., pressure) may be implemented, at least in part, by one structure 610 or by multiple structures 610.

Fig. 6A, 6B, 8A, 8B, 8C further illustrate examples in which illumination sources for dark field and/or bright field viewing may be provided by an illumination source 650 (such as, but not limited to, the annular light shown) disposed below the base support 620 to position the illumination device below the level of the cuvette 600. This design provides the headspace of the cuvette 600 to a pipette, sample processing device, or other device for unobstructed access to an opening or other structure located on an upper surface of the cuvette. Alternatively, some examples may position an illumination source 660 (shown in phantom) above the cuvette 600 to be used alone or in combination with a lower illumination source (e.g., lower illumination source 650 as shown). An objective lens 670 may be placed in the position shown, or in other designs, to view the illuminated sample. It should be appreciated that the use of relative motion between the cuvette 600 and the optical portions 650 and 670 may allow the system to view different analysis regions within the cuvette 600. Alternatively, only one of these structures is in motion to acquire data from a different region of the cuvette 600.

Referring now to FIG. 7A, one example of a suitable imaging system will be described in more detail. Figure 7A shows a schematic cross-sectional view of a different structure located below the base support 620. The cross-section is the area indicated by curved arrow 7 in fig. 6A.

Fig. 7A shows an example in which the cuvette 600 contains a base portion 606 and an analysis region 608 defined by a cover portion 612. Alternatively, the analysis region 608 may be defined within a single component. Alternatively, the analysis region 608 may be defined by using more than two components, such as, but not limited to, a separate covering component for each of the analysis regions 608. In one example, the laminate 606 comprises an optically clear plastic, such as, but not limited to, a cyclic olefin polymer insulating plastic for transporting premium optical components and applications. Some examples may be one or more layers or structures formed of glass, acrylic, clear polymer, or other transparent material. The cuvette 600 shown in FIG. 7A includes five separate analysis regions 608; the cross-section of these regions is shown in the figure; an analysis area 608 having such a cross-section may be rectangular, or square, or other shape. For example, the analysis region 608 may contain elongated channels that provide a shallow chamber with a relatively large surface area and through which the sample may be viewed. It should be understood that a cuvette 600 may include a single analysis region 608, or may include two analysis regions 608, or may include three analysis regions 608, or may include a dead number of analysis regions 608, or may include five (as shown in fig. 7A) or more analysis regions 608.

In this non-limiting example, the sample from which data is obtained may be limited to all or a portion of the area 608. In a non-limiting example, the optics below the base support 620 may include a ring light 650 including a toroidal reflector 652 and a light source 654. Other lighting components suitable for dark field lighting may be used; thus the optical system may include other illumination sources used alone or in combination with such an annular light. Some examples may use a mirror. Some examples may use a coated reflective surface. Some examples may use a different reflector than shown in the figures (e.g., a toroidal reflector may not be used when illuminating a sample). Some examples may use a parabolic reflector. Some examples may use a parabolic reflector in the shape of an elliptical paraboloid. Some examples may use multiple separate reflective sheets. Some examples may not use any reflector. Some examples may further obtain non-vertical illumination by using a light source angled from direct light, with or without assistance from one or more external reflectors.

The example shown in FIG. 7A shows excitation energy sources 680, 682, and 684, such as, but not limited to, laser diodes at specified wavelengths, mounted within a direct light source that illuminates the sample within the analysis region 608. In a non-limiting example, to facilitate compression of the package, the energy sources 680, 682, and 684 may emit light directly into a dichroic beamsplitter 690 (e.g., a dichroic beamsplitter or beam splitter) and then direct the activating wavelengths into the analysis region 608. The activation wavelength causes a fluorophore in a label, dye, and/or other substance in the sample to emit a fluorescent wavelength. The diverging fluorescence wavelength passes through the objective 670, through the dichroic beamsplitter 690, through an optical filter 692 into a detector 700, such as but not limited to a camera system. In a non-limiting example, dichroic beamsplitter 690 is designed to reflect the activating wavelength but pass the fluorescent wavelength and any desired wavelengths for optical viewing.

In one example, all of the fluorescence wavelengths illuminate the sample within the analysis region 608 simultaneously. For example, a detector 700 may be coupled to a programmed processor 710 that captures signals and/or images and resolves wavelengths associated with fluorophore emission fluorescence. In some examples, the excitation light source may illuminate the sample sequentially or using a fraction of the total number of excitation light sources. Of course, it should be understood that the system is not limited to fluorescence-based excitation of fluorophores within a sample, and that other detection techniques or excitation techniques may be used in place of or in combination with fluorescence. For example, some examples may also be used in conjunction with a fluorescence detector to collect darkfield illumination scatter information simultaneously or sequentially.

Light scattered by an object in a sample holder (e.g., a cell, or a ball, or a crystal) will be scattered from multiple angles, where a scattering angle may be measured and compared to incident light from a light source to the target. The plurality of scattering angles comprises a range of scattering angles. Such a sample holder may contain some of the structures shown herein and may be designed to provide access for internal light reflection. An objective lens designed for object imaging will collect and focus these reflected rays and then pass the rays to a detector. Such light focused by an objective lens and focused onto a detector may form a spot on the detector. In an example, the light transmitted from the objective lens to the detector may be focused by an additional lens; such focusing may reduce the size of the spot formed on the detector. The light focused onto a detector, whether or not through an additional lens, contains light scattered out of the object in the sample holder at multiple scattering angles.

The methods, systems, and apparatus (e.g., sample holders) shown herein may allow for a smaller range of scattering angles to be detected than other methods, systems, and apparatus, and thus may provide clearer and better quality images of specimens and objects within a sample. The cuvette design features shown herein may be used to control the angle and intensity of the incident light incident on the sample, for example by PIR and TIR, effectively controlling the angle of the scattered light being measured.

The scattering angle of light reaching a detector can be wider than ideal due to limitations imposed by many non-imaging optical systems (e.g., etendue, or the extent of light propagation through the system). For example, in some cases where toroidal light-cuvettes and LEDs are used in combination as the light source, light impinging on the sample may be spread at an angle of at least 20 degrees around the principal angle. In other words, if the chief ray strikes the sample at an angle of 60 degrees, other rays in the beam may strike the sample at scattering angles of about 50 to 70 degrees. It will be appreciated that the range of the cone of light scattering angles collected by an objective lens depends on the number of apertures of the lens. In this case, the light collected by the objective lens (e.g., a 40 degree numerical aperture) should be within a cone of about 30-70 degrees. As a result, scattered light over a wide range of scattering angles will reach the detector; for example, such a system would measure all light scattered by the sample focused within a large cone of about 60 degrees +/-40 degrees. However, as shown herein, some applications require detection of light within a narrow scattering angle, for example, within a very narrow angular range (60+/-5 degrees). In the application shown here, an aperture may be placed in the fourier (or back focal plane) of the objective lens (or any plane connected to it) in order to provide a measurement of light from this narrow range. In the fourier plane, the angular information is spatially encoded. Thus, depending on the shape and size of this aperture, certain angles of light from the sample may be prevented from reaching the detector (e.g., blocked or filtered out). An annular aperture will block or filter out the inner angles (like 60+/-30 degrees). The resulting measurement can be adjusted to the desired angle.

In one embodiment, an aperture may be provided so that light passes through the aperture after it exits an objective lens before contacting a detector. In one embodiment, an aperture may be provided such that light from an additional lens (after passing through an objective lens) passes through the aperture before contacting a detector. Wherein such an aperture is designed to limit the light reaching the detector, the light passing through the aperture will be reduced to a smaller scattering angle and smaller scattering angle than would be the case in the absence of a similar aperture. In an example, such an aperture may comprise a single hole, such as a circular hole. In an example, such an aperture may comprise a single annular hole, for example through which light passing through a circular ring may pass; while light passing through a central portion (e.g., a circle) cannot pass through. In an example, such an aperture may comprise two, or three, or more concentric annular apertures through which light passing through the annular ring may pass; it may also include a central portion (e.g., a circle) through which light does not pass. In examples, such an aperture may contain a shape other than a circle or ring.

Such an aperture, placed between an objective lens and a detector, for example between an additional lens and a detector through which light has passed an objective lens before passing, provides the advantage of accurate discrimination of scattered light from the sample, thereby improving the resolution of scattered light images (e.g. dark field images) obtained from the sample. In examples where light intensity may be a factor, the light intensity (e.g., from a light source, or multiple light sources) applied may be increased in a design having an aperture as shown herein as compared to a design without an aperture as shown herein.

A system may include a sample holder having the configuration shown and described herein, as well as a light source, dichroic mirror, and other elements shown in fig. 7A. As shown in fig. 7B, a system containing a similar configuration (e.g., similar to that shown in fig. 7A or other illustrations herein) may include a sample holder 600, a light source 650 (e.g., light source 654 or an excitation light source 680, or both), an objective 670, an aperture 694, an additional lens 696, and a fourier lens 698. An aperture 694 may have a single path that allows light to pass through to a detector 700. A detector 700 may be operatively coupled to a processor (e.g., a programmed processor) 710. In one example, an aperture 694 may have two paths that allow light to pass through to a detector 700. In one example, an aperture 694 may have three paths that allow light to pass through to a detector 700. In one example, an aperture 694 may have four or more paths that allow light to pass through to a detector 700. In one example, a passage in an aperture 694 may contain a circular hole to allow light to pass through to a detector 700. In one example, a passage in one aperture 694 may contain two, or three, or four, or more circular holes to allow light to pass through to one detector 700. In one example, a path within an aperture 694 may comprise a ring-shaped structure that allows light to pass through to a detector 700; and may include a central portion that does not allow light to pass through to a detector 700. In one example, a passage within one aperture 694 may contain two or more ring-shaped structures (e.g., concentric rings in one example), each of which allows light to pass through to one detector 700; and such an aperture 694 may include a central portion that does not allow light to pass through to a detector 700. Such a ring-shaped or several ring-shaped structures may have a circular, or oval, or other ring-shaped shape.

Thus, the system shown herein is for imaging a sample, comprising: a sample holder, a light source for illuminating objects in said sample holder, an objective lens for collecting and focusing scattered light from objects in said sample holder, said scattered light comprising light scattered at a plurality of scattering angles, an optical aperture for passing light from said objective lens, and an additional lens for focusing light from said objective lens onto said optical aperture, said optical aperture being designed to allow only a portion of light focused by said objective lens to pass through, said portion of light passing through said aperture consisting of scattered light at only a portion of said plurality of scattering angles.

As used herein, the terms "epi-illumination" and "epi-illumination" refer to illumination of a sample by a unidirectional line of light, which is directed generally away from an objective lens or other optical element used for observation or imaging of the sample. Thus, in the absence of fluorescence, an image of the sample illuminated by the epi-illumination is formed from reflected or scattered light from the sample (light traveling from the light source to the sample and then reflected or scattered by the sample onto the optical elements for observation, imaging or measurement). As used herein, the terms "transmission" and "transmission illumination" refer to illumination of a sample with light directed in a unidirectional direction, typically toward an objective lens or other optical element for observation or imaging of an object (light emanating from the light source and passing through the sample and then continuing to impinge on the optical element for observation, imaging or measurement). Thus, in the absence of fluorescence, an image of the sample illuminated by the transmitted illumination is formed from light passing through or scattered by the sample.

When a light source is located on the same side of a sample as the objective lens or other optical element used for object viewing or imaging, light from the light source is directed onto the sample, which is typically viewed or imaged by epi-illumination. However, a sample holder as shown herein can provide transmissive illumination for a sample in addition to epi-illumination, even when a single light source is located on the same side of a sample as the objective lens or optical element. Thus, the present system is able to provide bi-directional illumination without the need to place light sources on both sides of a sample at the same time. Such a design is very compact, saving resources; and since the light source and other optical elements are located on one side of the sample holder, this design allows unobstructed access to one side of the sample holder without interference from the optical elements. Such a design therefore provides the advantage of being able to load, mix and remove a sample and reagents within the sample holder without interfering with the optical imaging or measurement, or with the devices and elements used for optical imaging and measurement.

As shown in the images of fig. 4A and 4B, the addition of transmitted illumination to the dark field image greatly enhances the image effect and greatly improves the information that can be acquired from the image. The methods and systems shown herein provide images with greatly enhanced results by illumination from a single direction and a single light source through a combination of both epi-illumination and transmission illumination.

As shown herein, a sample holder such as a cuvette 600 (e.g., as shown in FIGS. 8A-8D) is designed to allow for internal reflection of light (PIR or TIR) from a light source such that a sample retained within an analysis region 608 of a cuvette 600 can be illuminated by direct light (epi-illumination; e.g., light traveling along pathway 830) or indirectly reflected light (transmission illumination; e.g., light traveling along pathway 820 or 825). As shown here, light from a light source that is exposed on the same side of a cuvette 600 as the optical elements 670, 690, 700, etc., may provide both epi-illumination and transmission illumination for a sample.

A further example will now be described with reference to fig. 8A-8D. Fig. 8A-8D show a schematic cross-sectional view of a cuvette 600 and a portion of the dark-field diffuse light source such as, but not limited to, the annular light 650 shown in fig. 6A and 6B. Base support 620 is also shown in FIGS. 8A-8D. FIGS. 8A-8D include brackets and arrows to indicate structures or portions of structures; for example, the parenthesized designation 600 indicates the entire cuvette 600 shown in the figure; the bracket labeled 612 indicates the covered portion 612 of the cuvette 600. Arrows 621 to 626 in fig. 8A indicate the size of the indicated portion of the cover portion 612. It should be understood that these dimensions may vary in different instances of a cuvette 600, and that these variations may depend on size, application, materials, optical wavelengths, sample, and other elements and factors related to the structure and use of a cuvette 600. For example, the distance 621 between the support structures 604 may be between about 0.1 millimeters (mm) and 1 centimeter (cm) in an example, and may be between about 1mm and 100mm, or between about 1.5mm and 50mm, or between about 2mm and 20mm in an example. In further examples, the distance 621 between the support structures 604 may be between about 0.5mm and 10mm, or between about 1mm and 5 mm. In examples, the height 622 of a support structure 604 may be between about 0.1mm and 100mm, or may be between about 0.5mm and 500mm, or between about 1mm and 25 mm. In further examples, the height 622 of the support structure 604 may be between about 0.1mm and 10mm, or may be between about 1mm and 5mm likewise, in examples, the height 623 of one thickness control region 613 may be between about 0.1mm and 100mm, or between about 0.5mm and 50mm, or between about 1mm and 25 mm. In further examples, the height 623 of one thickness control region 613 may be between about 0.1mm to 10mm, or between about 1mm to 5 mm. In examples, the thickness 624 of one layer 800 may be between about 0.01mm and 10mm, or between about 0.05mm and 1mm, or between about 0.1mm and 0.5 mm. In examples, a width 625 of one analysis region 608 may be between about 0.05mm to 100mm, or between about 0.5mm to 50mm, or between about 1mm to 25 mm. In further examples, a width 625 of one analysis region 608 may be between about 0.1mm to 10mm, or between about 1mm to 5 mm. In examples, a width 626 of a support structure 604 may be between about 0.1mm to 100mm, or between about 0.5mm to 50mm, or between about 1mm to 25 mm. In further examples, a width 626 of a support structure 604 may be between about 0.05mm to 10mm, or between about 0.5mm to 5 mm.

It is to be understood that optical components and arrangements for illumination, activation, observation of emission wavelengths, and similar functions shown in one illustrative figure may be illustrative of components and arrangements for other illustrative examples, even if the particular components or illustrations are not explicitly shown in the respective illustrative figures. For example, although an annular light 650 or other illumination source 650 is not included in fig. 8D, in any of the illustrated examples, as well as others, an annular light 650 or other illumination source 650 (see fig. 8A, 8B, and 8C) may be used to illuminate the analysis region 608 (analysis region 608 is shown in fig. 8A and 8B). As exemplary of the optical components suitable for use with one cuvette 600, annular light components 652 and 654 are shown in FIGS. 8A, 8B and 8D; in an example, other lighting components or other numbers of lighting components may be used. For example, the light source 654 may be white light or some light source such as, but not limited to, a Light Emitting Diode (LED) or laser diode having a particular output wavelength or output range. Alternatively, the ring light source 654 may be a fiber optic cable (e.g., with a plurality of splices) designed to provide a ring light. Alternatively, the light source 654 may be an LED with a specific narrow divergence angle controlled by the reflector. It may be very desirable to control the angle of divergence from one annulus of light by the choice of light source and/or by the design of the reflector.

In a non-limiting example, a light source 654 may use laser illumination to provide a narrow light pattern, resulting in a lower transmitted illumination background (illumination components all on one side of the sample) in the presence of epi-illumination, because the light source: providing a narrow spot of light (directed into the sample analysis region); providing light of a narrow spectral width (e.g., the wavelength of the light lies within a narrow range centered on a particular dominant wavelength); and it is a continuous light source. Alternatively, using an LED as the illumination source 654 may also provide a small spot (e.g., a small spot within an analysis region 608), which may also provide some of the beneficial characteristics of a laser source. For these reasons or others, a laser light source (or an LED, providing a small spot)) can effectively reduce the background signal level compared to other illumination designs. A laser light source may reduce scattered light that is typically present in a former compared to a more diffuse light source, which may reduce background light within a channel (e.g., within a sub-first analysis region 608) by reducing light scattered into the channel from an adjacent channel (e.g., from an adjacent second analysis region 608). Such that the laser light source may create less transmissive illumination background than would be expected from diffuse light source illumination. It is of course desirable that the reduction in transmitted illumination is less than the reduction in background illumination, and a significant drop in background light source can result in a more distinguishable signal. Optionally, an LED is used as the illumination source 654 to provide a diffuse light pattern to increase background and transmission illumination. It is of course desirable that the increase in transmitted illumination is greater than the increase in background illumination.

Some examples of cuvettes may include cuvettes formed from multiple separate layers bonded together, cuvettes molded from one or more materials, and/or addition of reflective layers on different surfaces of the cuvettes to enhance single or multiple internal reflections (e.g., increase TIR or PIR).

In an example where the system, cuvette, and optical elements shown herein may be used in conjunction with fluorescence, we may desire dark field illumination for use with such systems and cuvettes, rather than white light illumination. However, some instances may be where white light illumination is desired, for example, if fluorescence detection cannot be used in conjunction with a darkfield microscope and/or a brightfield microscope.

Fig. 8A and 8B show that in some instances the device may have some optically non-transmissive layer, such as layer 800, within the cuvette 600. This may be useful in some instances where the light source 654 is diffuse and light is not directed to a particular location. The layer 800 can block light that does not enter the cuvette 600 at a desired angle and/or position. The sheet 800 may be designed to be placed in a position to prevent illumination except through the area below the analysis region 618. Some instances may have only certain regions closest to the analysis region 608 masked. Some examples may have more than one layer covered or non-transmissive material. Some examples may be masked or used with non-transmissive materials in different orientations, such as but not limited to one being horizontal and the other being vertical or non-horizontal.

It should be understood that in some instances, one layer 800 may be optically transmissive. For example, fig. 8D shows an example in which one layer 800 is optically transmissive. In some examples, a layer 800 may comprise an optically transmissive material having a refractive index that is different from the refractive index of a thickness control region 613 and/or a base support 620. In some examples, a layer 800 may comprise an optically transmissive material having a refractive index that is the same as the refractive index of a thickness control region 613 and/or a base support 620.

8A, 8B, and 8C, a light source is positioned below one of the cuvettes 600 (proximate optics 652 and 654) and provides light directly from below the base portion 606. Such a light source may be more readily understood by the demonstration shown in fig. 8D. As shown in these figures, a light source 650 may include a toroidal light 654 and a toroidal reflector 652. Other elements, including but not limited to lenses, filters, gratings, lenses and other reflective surfaces, optical fibers, prisms, and other elements may be included. In one example, a light source may comprise a laser, or an LED, or other light source; and may include a light source that transmits light from such a light source to another location and/or directs light directly to an optical element. One design criterion for an optical system is the dispersion or divergence angle of the light rays emanating from the light source; within a given distance of the light source, a beam of width D with low dispersion provides a smaller spot than a beam of width D with high dispersion. Typically, a light source 650 that provides light of low dispersion is preferred, such optical elements and structures may be designed to provide substantially parallel light, e.g., with most or all of the light directed along substantially parallel lines toward the sample (e.g., toward an analysis region 608). However, there are some examples of preferred diffuse or diffuse light, and a light source 650 with high dispersion may be used.

As shown in fig. 8C, an example of an optical system suitable as part of the apparatus or system shown herein may include optical elements (e.g., a light source 650, an annular light 654 as shown in fig. 8C, and an objective lens 670), a cuvette 600, and a base support 620 designed to hold and position an imaging cuvette. In the example shown in fig. 8C, a base support 620 may include an optical arrangement 802 to reflect (or diffract, or alter the path of) light emitted from a light source 650. As shown in fig. 8C, the optical arrangement 802 may include a microlens array. It should be understood that the optical arrangement 802 may contain any suitable optical structure. In an example, the optical configuration 802 may contain micro-lenses, or diffraction gratings, or Fresnel lenses, or convex lenses, or concave lenses, or other shapes and structures that may reflect, diffract, or alter the line, or combination of effects, on light. In an example, such an optical configuration 802 may contain a different material than the base support 620, and a different index of reflection than the rain base support 620. For example, light affected by the optical arrangement 802 may be suitable for being directed to an analysis region 608, either directly or indirectly through reflection (e.g., internal reflection) using the methods shown herein, such that both epi-illumination and transmission illumination are provided to a sample within an analysis region 608. As shown in the example of fig. 8C, this example may also include an optical path that bypasses the optical arrangement 802. Such an optical path may be better suited for imaging a sample within an analysis region 608 than imaging using an optical arrangement 802 path. In an example, both types of optical paths (e.g., bypassing the optical arrangement 802 and passing through the optical arrangement 802) may be provided simultaneously, which may provide suitable optical conditions for image analysis of a sample illuminated both epi-and transmission by a light source located on the same side of a cuvette 600 as the light source 650.

The cuvette 600 includes structures that affect the path of light that strikes the cuvette and the sample within the cuvette. Such transmitted illumination may be affected by light reflected (e.g., internal reflection) within a cuvette 600, including or primarily by Partial Internal Reflection (PIR) or Total Internal Reflection (TIR) from, for example, a surface 612, or other surface or associated surfaces. Other examples of ray paths for performing TIR are shown, for example, in fig. 8A, 8B, and 8D.

As shown in FIG. 8D, in one example, a cuvette 600 in an optical system of an apparatus or system shown herein and suitable for use with the methods shown herein may include structures that affect the path of light that strikes the interior of the cuvette 600, such as light that strikes an analysis region 608 and a sample within an analysis region 608 of the cuvette 600. As shown in fig. 8D, a layer 800 may include structures that reflect, diffract, or otherwise affect or alter the path of light into an analysis region 608. This change in optical path may have an effect or increase on the illumination of the sample within one of the analysis regions 608. In the example shown in FIG. 8D, light is intended to enter the level 800 from a lateral direction; the light path is altered by the shape (and material properties) of the layer 800 and enters the analysis region 608 in a desired direction. For example, an outer surface of a layer 800 may be planar (e.g., the outer surface 674), or may be curved (e.g., the outer surface 676). For example, an inner surface of a layer 800 may be planar (not shown in fig. 8D; see similar surfaces in fig. 8A and 8B; although the layer 800 in fig. 8A and 8B is not optically transmissive, these surfaces are shown as planar), or may be curved (e.g., inner surface 678 shown in fig. 8D). In one example, this change in optical path is very effective for providing both epi-illumination and transmission illumination to the sample in one analysis region 608.

Figures 8A, 8B, 8C, and 8D show light paths within a sample holder, providing an example of TIR and PIR generation at an upper surface 614 within a cover portion 612 and/or at a surface 618 of a support structure 604. A sample holder, such as a cuvette 600, may contain an optically transmissive surface through which light may pass; in one example, such an optically transmissive surface may allow light to pass therethrough without significant distortion or intensity reduction. A sample holder, such as a cuvette 600, may be made of an optically transmissive material, with the effect that light may enter the sample holder. In an example where a sample holder is made at least in part of an optically transmissive material, light may penetrate an optically transmissive surface of a sample holder and may travel within the sample holder. In one example, light traveling within a sample rack may be reflected at one or more surfaces and travel along a reflected path within a sample rack. When light enters a sample holder from a light source located outside the sample holder through an optically transmissive surface of the sample holder, the light may travel within the sample holder in a direction away from the light source; may also be reflected by a surface of the sample holder, such that the reflected light, after being reflected, may travel in a direction facing the light source. This reflection may be PIR or TIR.

Thus, light traveling within a cuvette 600 may be reflected by a surface (e.g., a surface 614 or 618). This reflection may be very effective for indirect illumination of a sample within an analysis region 608; after use in combination with direct illumination (light not reflected before it impinges on a sample), the sample may in this way receive epi-illumination (illumination from the same side of the optical detection element) and transmission illumination (illumination from the opposite side of the optical detection element).

It should be understood that the light wavelength, material, surface, and design to enhance or enhance the PIR may not be suitable or effective to enhance or enhance TIR. It is understood that light wavelengths, materials, surfaces, and designs that enhance or enhance TIR may not be suitable or effective for enhancing or enhancing PIR. Thus, there are some designs and structures that may contribute to one of PIR and TIR, but not the other. In the example, there are some designs and structures that may contribute to both PIR and TIR. In the example, there are some designs and structures that may not contribute to both PIR and TIR.

As shown in fig. 8A, the support structure 604 may have a rectangular or square cross-section. It should be understood that a support structure 604 may have more than a square or rectangular cross-section; for example, as shown in FIG. 8B, a support structure 604 may have a triangular cross-section; other cross-sectional shapes (e.g., circular or semi-circular, or serrated, or irregular) may also be suitable for use in the systems and cuvettes shown herein. PIR and TIR are adjustable characteristics that may be selected based on the material used for the cuvette 600, any coatings, cladding, or coverings used, and the geometry and/or thickness of the thickness control region 613 of the cuvette 600. In one example, PIRs may be preferred, and light, materials, and design structures may be selected to enhance PIRs.

In an example, TIR may be preferred. In an example, the wavelength of light or wavelengths of light from one light source 650 may be selected to enhance TIR. In an example, the material, thickness, surface, design, and other structures of a cuvette 600 may be selected to enhance TIR. For example, the height of thickness control region 613 (measured from the bottom of cover portion 612 to level 800) may affect the angle and intensity of light reflected by TIR reaching analysis region 608. A cuvette 600 design that allows TIR of light within the cuvette to allow for oblique illumination of a sample (illumination from above the sample) is desirable, particularly for dark field microscopes. In some examples, it is desirable to maximize TIR from the sample. Alternatively, in some instances, a cuvette 600 may be designed to provide TIR only from surfaces above the analysis region 608. Alternatively, some examples may be designed to provide TIR only from surfaces above the thickness control region 613 (e.g., the examples shown in fig. 8A and 8B, generally above the analysis region 608). Alternatively, in some examples, a cuvette 600 may be designed to provide TIR from other surfaces of the cuvette 600; for example, TIR light from other surfaces of the cuvette 600 may be provided to scatter light at an oblique angle, with the effect that the light is reflected directly back into the analysis region 608. .

The design and materials used to construct a cuvette 600 may be selected and designed to provide light TIR. For example, in some instances, designs that provide TIR or increase or enhance the amount of TIR include, but are not limited to: the dimensions of thickness control region 613 are compatible with or can enhance TIR; the angle of one surface 614 or one surface 618 (e.g., as compared to the incident light ray) is compatible with or can enhance TIR; the shape, texture, or coating of one surface 614 or one surface 618 is compatible with or can enhance TIR; the difference between the index of reflection of the material from which one thickness control region 613 is made and the index of reflection of the material and space of surface 614 bounding one thickness control region 613 is compatible with or can enhance TIR; the difference between the index of reflection of the material from which a support structure 604 is made and the index of reflection of the material and space of surface 618 bounding a support structure 604 is compatible with or can enhance TIR; and other structures and designs. To enhance TIR, the first material within which the light is reflected (internally) should contain a higher index than the second material through which the light would pass if it were not internally reflected; since a similar second material, typically air, has a refractive index close to 1, it is not difficult to ensure a high refractive index. To improve TIR, the angle of incidence must be greater than the critical angle. For example, referring to the example shown in FIG. 8, the material forming thickness control region 613 and support structure 604 (e.g., the outer portions of surfaces 614 and 618) should have a refractive index greater than air. In one example where TIR is desired in a layer 800, the material of layer 800 should have a lower refractive index than the material of thickness control region 613 to ensure TIR occurs in the walls shown in fig. 8A, 8B, and 8D. In another example, a layer 800 of material having a higher refractive index than the material of the thickness control regions 613 may produce TIR at the boundary (between a layer 800 and a thickness control region 613) with the effect that the angle and material may be adjusted to optimize the composition of transmitted illumination directed at a sample within an analysis region 608.

In an example, one surface 614 or 618 may be coated or treated to affect or reduce the reflection (PIR or TIR) of that surface. In an example, one surface 614 or 618 may be coated or treated to reduce light escaping from the surface. For example, even if one surface 614 or 618 is suitable for TIR, or the amount of TIR is increased, some light rays may be transmitted or reflected by that surface 614 or 618. An optically absorbing coating or material may be disposed on or applied to such surface 614 or 618, or a portion thereof, to reduce the amount of stray light escaping from a cuvette 600. Such a light absorbing coating may be, for example, a dye, an ink, a paint, a surface treatment, a colored tape, or other coating or surface treatment technique. In an example, a black or other light absorbing solid material may be applied against or in close proximity to a surface 614 or 618 to provide an optically absorbing surface.

Alternatively, in some examples, a cuvette 600 may be designed such that one or more portions of the cuvette provide no TIR light (or only an insignificant amount of TIR) or no PIR light (or only an insignificant amount of PIR). For example, in some instances, a cuvette 600 may be designed to provide no TIR or PIR light (or only an insignificant amount of TIR or PIR) from the support structure 604. Alternatively, in some instances, a cuvette 600 may be designed to provide no TIR or PIR light (or only an insignificant amount of TIR or PIR) from a surface 618. Designs that do not provide TIR or PIR light, or provide only an insignificant amount of TIR or PIR, include, but are not limited to: the thickness control region 613 is not dimensioned to fit or enhance TIR or PIR; one surface 614 or one surface 618 (e.g., as compared to the incident light ray) is at an angle that is not suitable for or does not enhance TIR or PIR; the shape, texture, or coating of one surface 614 or one surface 618 is not suitable or does not enhance TIR or PIR; the difference between the index of refraction of the material from which a thickness control region 613 is made and the index of refraction of the material and space of surface 614 bounding a thickness control region 613 is not suitable or does not enhance TIR or PIR; the difference between the index of reflection of the material from which a support structure 604 is made and the index of reflection of the material and space of surface 618 bounding a support structure 604 is not suitable or does not enhance TIR or PIR; and other structures and designs.

Alternatively, in some examples, a reflective material may be disposed on or attached to one surface 614 and/or one surface 618. Such a reflective material may be, for example, a metal, such as silver, or only, or aluminum; may be a dielectric such as magnesium fluoride or calcium fluoride, or other oxide salts or metals; or other reflective material. Typically, such a reflective coating may be very thin (e.g., may be less than 0.1 microns, or may be 100 microns thick at maximum). Alternatively, a reflective material (e.g., a reflective coating) may be merely placed on or affixed to surface 614. Alternatively, a reflective material may be simply placed on or attached to surface 618. Alternatively, the surface 618 may be treated to be black so as to have light absorption properties. In another example, one surface 614 may be treated to be black to have light absorption. Some examples may choose that the thickness control region 613 is wider than the analysis region 608. For some practical laser illumination examples, the layer 800 may be removed or referred to as light transmissive because the laser illumination has given sufficient focus so that no dark regions are required between the analysis regions 618.

In a non-limiting example, rays may also be made to travel along path 820 from the vicinity directly into the analysis region 608 using PIR and/or TIR. 8A, 8B, and 8D, light traveling along path 820 is reflected toward analysis area 608; while light traveling along path 825 will undergo multiple reflections as it travels within cuvette 600 and eventually reaches analysis region 608. As shown in FIG. 8B, light traveling along path 820 undergoes multiple reflections as it travels within cuvette 600, ultimately reaching analysis region 608. This reflection may be a PIR, as shown in fig. 8B, or may be TIR. Illumination shown by rays following paths 820 and 825 in FIG. 8A and illumination shown by rays following path 820 in FIG. 8B are transmitted illumination, as is conventional. The illumination shown in FIGS. 8A and 8B by rays traveling along path 830 shows rays directly from the ring of light rather than by TIR: this is epi-illumination. The combined use of two types of light components from a light source below the sample (or on only one side of the sample) provides improved functionality over the use of only one of the light components as a light source. This is particularly useful for dark field microscopy.

One non-limiting example of an application of the examples shown in figures 8A-8D is to measure the scattering properties of cells in the sample using dark field illumination. Dark field microscopy is a well-established method, used primarily as a contrast-enhancement technique. In dark field microscopy, the entire imaged background is completely dark, since only light scattered or reflected by the sample is imaged. High quality dark field microscopic techniques do not measure the scattering properties of cells using the traditional "side scatter" parameters in flow cytometry.

In hardware, illumination for dark field microscopy requires oblique angle illumination, e.g., all light from the illumination source should first contact the sample and then enter the objective lens. In a non-limiting demonstration, the illumination wavelength should not activate any other fluorophores already present in the sample. Alternatively, such illumination allows imaging using a high Numerical Aperture (NA) lens. In a non-limiting example, the NA value of a conventional lens aperture size associated with an optical microscope may be at least 0.3. Alternatively, the NA value is at least 0.4. Alternatively, the NA value is at least 0.5. Alternatively, some examples may use oil immersion objective lenses to achieve a desired NA value, particularly when the lens aperture value is limited below a certain level.

A conventional approach for dark field illumination is to use transmission illumination, where the sample is located between the imaging lens and the dark field light source. Thus, with this conventional arrangement, the detector and the illumination component are not on the same side of the sample. Conventional epi-illumination methods (imaging lens/objective and the light source on the same side of the sample) require the use of a specially manufactured objective and typically do not allow the use of the NA objective, which limits the functionality of the overall system.

In contrast, at least some of the dark field illumination system examples described herein have the following attributes. In terms of hardware, the schematic diagrams of the examples in fig. 8A-8D are "epi-down", where the annular light for dark field illumination is located on the same side of the sample as the objective lens. This design is highly desirable from a system perspective, although other embodiments with light sources on opposite sides may be used alone or in combination with the embodiments described herein. In one non-limiting example, the ring light is designed such that its LEDs and/or the laser light source 654 all lie in the same plane and point in the same direction (light source in the same horizontal plane, light pointing up). In some examples, the light rays may lie in the same plane, but the pointing directions are non-parallel, such as but not limited to a pyramidal pattern. In some instances the light rays may lie in different planes, but the pointing directions are uniform. In some examples, the light rays may be in different planes and the pointing directions are also non-parallel, such as but not limited to a pyramidal pattern. In some examples, the light is reflected off of a ring mirror 652 to confound tilt illumination of the sample.

In addition to the optical characteristics of the annular light and the annular reflector, the optical characteristics of the cuvette 600 shown in the example of fig. 8A-8D also significantly affect the dark field illumination. In this example, the cytometer cuvette 600 is designed such that light from the ring light 650 falls directly on the sample; but in addition, light will also "reflect" onto the sample via some structure of the cuvette, to emulate "transmitted" illumination. This reflection may be in TIR mode and/or in true reflection mode.

Note that any transmitted illumination design allows us to measure forward scattered light from a sample; while an epi-illumination design only allows us to measure the backscattered light from the sample. The forward scattered light is typically two orders of magnitude higher in intensity than the backward scattered light. Thus, the use of transmitted illumination allows us to use very low illumination intensities, reducing the side effects that are detrimental to the sample.

As an example shown in FIG. 8A, the ring light 650 (or other illumination source) and cuvette 600 provide a system that can be adjusted where the intensity of the transmitted and epi-illumination can be adjusted to improve its performance over conventional transmitted illumination. Similarly, in the example of FIG. 8B, the ring light 650 (or other illumination source) and cuvette 600 provide a system that can be adjusted so that the intensity of the transmitted and epi-illumination can be adjusted to improve its performance over conventional transmitted illumination. Such tuning can be accomplished by the advantages of the materials chosen (e.g., their optical properties) and the geometric design of the cuvette to control the angle and extent of total internal reflection.

As shown in fig. 8C, the structure 802 may alter the path of incident light and may be used to enhance both transmission and epi-illumination. As shown in fig. 8D, the shapes and designs of the surfaces 674, 676, and 678 may alter the path of incident light (e.g., lateral illumination) and may be used to provide or enhance both transmissive illumination and/or epi-illumination.

Fig. 8E provides a schematic representation of the transport of a cuvette 600 from a sample preparation position to a sample viewing position near an optical detector D. As indicated in the figure, a sample holder 600 may be moved from a position to a position near or onto a detector D. A detector D may include a stage for receiving, retaining, and positioning a cuvette 600. Samples may be added to the sample holder through the inlet 602 (e.g., 6 inlets 602 in the example shown in fig. 8E) and may be positioned for optical observation and measurement within an analysis region 608 (not shown, as an interior surface of a support structure 604) of the cuvette 600 shown in fig. 8E. The sample remaining in one of the analysis regions 608 may be illuminated and may be detected by one of the detectors D. In an example, one detector D may be designed for high quality viewing or imaging.

One detector D shown in fig. 8E may contain one cell counting unit or cell counting module; or may itself be part of a cell counting unit or module. Such a cell counting unit or module may comprise a separate unit or module for sample analysis. In an example, other analysis functions and devices may be included in one detector D; or packaged with a detector D; or may be designed for use in conjunction with a detector D. In an example, a system for sample analysis as described herein may include such a cell counting unit or cell counting module, e.g., including a detector D for analyzing a sample in a cuvette 600. In an example, a system for sample analysis described herein may contain such a cell counting unit or cell counting module; and contains, in addition to a detector D for analyzing a sample in a cuvette 600, other units or modules that provide other analytical functions and devices. In such a system, such other units or modules may be packaged with one detector D; or may be designed for use in conjunction with a detector D. Such other analysis functions and devices may be applied to a sample; for example, the analysis functionality and apparatus may be used to analyze a sample or a portion of a sample present in a cuvette 600. In examples, such analytical functions and devices may be used to analyze a different portion of the sample present in a cuvette 600 (e.g., a sample may be split into two or three portions, one of which is placed in a cuvette 600 for cytometric analysis, and one or more of which is packaged with, located near, or otherwise associated with a cytometric unit or module). Thus, for example, a sample (or a portion of a sample) may be measured and/or analyzed in a chemical analysis unit, or a nucleic acid analysis unit, or a protein analysis unit (a unit for analyzing a sample using antibodies or other specific binding molecules), or other similar units or combinations of units, independently of the analysis performed by such a cytometry module. Such analysis may include analysis of small molecules and elements expressed within a sample (e.g., by a common chemical unit); analyzing (e.g., by a nucleic acid unit) nucleic acid molecules expressed within a sample; analyzing (e.g., by an enzyme-linked immunosorbent assay (ELISA) unit) the expressed protein and/or antibody-reactive antigen within a sample; or a combination of several assays. In addition, the system as shown in FIG. 8E and discussed herein may include a controller to control and schedule operations within one or more units or modules.

Fig. 8E provides a schematic illustration of further details of the system, including a transport mechanism for transporting a cuvette from a sample preparation position to a sample viewing position near an optical detector D. A system, such as the one example system shown in fig. 8F, may include a plurality of sample analysis modules that may be designed to operate independently; or in some instances are designed to cooperate with each other. The system shown in FIG. 8F includes a single cell counting unit 707 with a single detector D; in an example, a sample (or a portion of a sample) analyzed in any or all of the analysis units 701, 702, 703, 704, 705, and 706 may be conveyed to the cytometry module 707 for observation and measurement by the detector D. Independently of the analysis performed by the cytometry module 707, a sample (or a portion of a sample) may be measured and/or analyzed within a chemical analysis unit 715. Such analysis performed in a chemical analysis unit 715 may include analysis of small molecules and elements expressed in a sample (e.g., by a common chemical unit); analyzing (e.g., by a nucleic acid unit) nucleic acid molecules expressed within a sample; analyzing (e.g., by an enzyme-linked immunosorbent assay (ELISA) unit) the expressed protein and/or antibody-reactive antigen within a sample; or a combination of several assays.

The system shown in fig. 8F may include a controller to control and schedule the operations within one or more of the modules 701 and 707. The sample may be loaded onto other elements of the sample holder and analyzed in the exemplary system shown in fig. 8E. Such a system, or modules within such a system, includes, for example, a sample processing system 708, pipettes for taking, moving, and dispensing samples, including aspirant and positive displacement pipettes 711, 712, centrifuges 713, spectrophotometers 714, chemical analysis units 715, photomultiplier tubes (PMTs)716, cassettes 717 for holding disposables and tools, such as pipette tips and other tips, and other components. The modules and other components may be supported by a rack 709 or other support structure. Samples, disposables, tools, and other elements may be transferred within a module, and may be transferred between modules (e.g., between a module 701-706 and a cytometry module 707).

Fig. 8E and 8F show that the sample holder, such as cuvette 600, may be transported from one location (e.g., where sample processing may be performed) to another location (e.g., detector D shown in fig. 8E and 8F). The cuvette 600 does not release liquid into or onto the detector D, but rather is a self-contained unit that retains all of the sample therein. There may be one or more, two or more, three or more locations on or near the detector D that contain transparent surfaces to which the cuvettes 600 or other sample holders may engage, providing a transparent interface for detection of sample signals. The elements shown in fig. 8F, as well as further illustrations of such elements and their applications, can be found in U.S. patent application serial No. 13/769,779, which is incorporated herein by reference in its entirety.

Dark field

At least some examples herein include a dark field illumination source and a cuvette. The relevant structure of the cuvette 600 is related to the design of the cuvette dimensions, optical materials and geometry. The cuvette increases the degree of dark field illumination by reflection (e.g., by TIR and/or PIR). In one example, the system may use both transmissive and epi-darkfield illumination for one sample.

In some examples shown herein, the cuvette 600 used in conjunction with the light source 650 may allow for both transmission and epi-illumination to use a physical system in the epi-configuration (e.g., the light source is on the same side of the sample as the objective lens). The most basic cuvettes are designed for receiving and viewing biological samples. In an example, the cover portion 612 may have a particular design. It is known that different materials may have different indices of reflection; a material having a desired index of reflection may be selected to produce a cover portion 612, or a base support 620, or other elements and components of a cuvette 600 and its associated components and constituents. For example, in some instances, one cover portion 612 or one base support 620 may be made of glass. For example, in some instances, one cover portion 612 or one base support 620 may be made of quartz. For example, in some examples, a cover portion 612 or a substrate support portion 620 may be made of an acrylic plastic, or a colorless polymer (e.g., a cyclic olefin copolymer, a polycarbonate, a polystyrene, a polyethylene, a polyurethane, a polyvinyl chloride, or other polymer or composite polymer), or other transparent material.

We can design the material of the upper cover portion 612 to facilitate illumination and image collection. In examples, to illuminate a sample, the light source 650 may be a ring light 650 (e.g., may be ring shaped) may have light sources 654 placed in an intermittent or continuous manner, and a curved reflector 652 may be used to direct light toward the sample.

In dark field microscopy, the sample is illuminated by an oblique light. In dark field microscopy, light entering the microscope optics is lightly scattered by the sample, allowing measurement of the scattering properties of cells, particles and other materials and structures in the sample. The dark field image is black if no cells, particles, structures or other substances are present in the sample.

In a non-limiting example, the reflector 652 and the LED654 of the ring light 650 are designed to reflect light such that a small portion of the light returns directly to the objective lens as non-specific background. The system is designed to return light directly into the analysis region 608 by TIR at the cuvette surface. Light rays reflected from a surface, whether TIR or other types of reflection, are directed for illumination of a sample within the analysis region 608. Cells, particles, and structures within the sample in the analysis region 608 receive light directly from the annulus below the cells (e.g., via epi-illumination). Also as shown here, light from the upper surface (reflected) is also directed to the analysis region 608 (e.g., by transmission illumination).

Thus, according to the systems and methods described herein, at the same location as the annulus light 650, light may be directed from a single annulus light source to the analysis region 608 from two directions (epi-illumination and transmission illumination). In the example the illumination is all tilt angle illumination. We can control the relative intensity of the two light components by the design of the cuvette and the materials used for the cuvette.

This dark field illumination is different from conventional dark fields. For example, in the example shown here, the dark field illumination is provided by light reflected by TIR from a cuvette surface. By way of non-limiting example, in one example, a system shown herein may use a reflective layer on the back side of a particular surface of the cover portion 612 to reflect all light. By way of non-limiting example, in one example, a system shown herein may use a reflective layer on the back side of a particular surface of a cuvette 600 to reflect all light. Some examples may use a totally or selectively reflective background.

For example, in an example, it is desirable to emit light at an oblique angle to maintain illumination of a dark field. In some examples, the light source 654 may emit light at an oblique angle, and thus the reflector 652 may not be needed or may not be used the reflector 652 may improve the quality of the light source 654 because all of the light rays are in the same plane and are emitted in the same direction. Alternatively, the angled light source 654 may be used in place of or in conjunction with a reflector.

It should be appreciated that even though the intensity of the light of the transmitted illumination component of the illumination may be less, such as 10 times, than the intensity of the corresponding epi-illumination, the scattered light from the transmitted illumination in the cells or other material in the sample may be 200 times more intense than the scattered light from the epi-illumination. That is, the intensity of scattered light from a quantity of epi-illumination may be 200 times stronger than the intensity of scattered light from the same quantity of transmitted illumination in cells or other material within the sample resulting from the transmitted illumination. Thus, a small amount of transmitted illumination can significantly enhance light scattering by the cell.

When using epi-illumination only, the light collected by one objective is only the light reflected by one sample. However, diffraction is an important component of scattering, and the use of transmissive illumination may provide a certain amount of diffracted light (e.g., light diffracted by the sample). However, the light collected from the epi-illumination does not include the diffracted light of the sample (no light is reflected back to the light source after diffraction). Thus, when using both transmission and epi-illumination, one objective lens collects light with reflected, refracted and diffracted components. The traditional approach is to use the transmitted dark field illumination entirely, rather than requiring the placement of the optics components on both sides of the sample, and therefore the space occupation on the design is very significant. In contrast, the systems and methods described herein can provide both epi-illumination and transmission illumination using only epi-illumination optics alone. The examples shown herein may provide the benefits of both epi-illumination and transmission illumination to the sample while achieving the space saving effects of epi-illumination designs.

Designing the sample holder and light source together may allow an epi-illumination configuration to increase the amount of transmitted illumination of the sample, particularly possibly providing consistent transmitted illumination. Some examples may use a light reflecting plane. Some examples use TIR, which can be adjusted to produce the desired transmissive illumination, including uniform and at an oblique angle into the analysis region 608 for dark field illumination of the sample. A cuvette 600 may be designed to provide transmissive illumination for an analysis region 608 using reflection, for example using TIR and/or PIR, from only one source of epi-illumination within the structure. In a non-limiting example, a thicker cover portion 612 allows light to be reflected back into the target area 608 via TIR (or PIR; or both). Moreover, the systems and methods shown herein not only provide light rays that originate from TIR (or PIR; or both) back into an analysis region 608, but are uniform in the light rays that return into an analysis region 608. The examples in fig. 8A, 8B and 8D contain specific surfaces, specific black surfaces and specific reflective surfaces that are present at an angle such that the uniform return of light to an analysis region 608 can effectively provide uniform transmitted illumination for a sample within an analysis region. Alternatively, we can place a fully reflective surface on top (such as but not limited to a planar cover portion 612 as shown in fig. 7A and 7B, or alternatively cover selected areas over area 613 as shown in fig. 8A, 8B and 8C). In contrast, light traveling in conventional hardware may undergo some reflection, including perhaps some TIR (or PIR, or both), but the light may not return into the region 608.

In a non-limiting example, rather than using a high complexity, high cost system such as may contain 16 laser light sources, the examples shown herein develop a more integrated detection system for imaging and identifying different cells and types within a sample.

Combining all of these different types of information together, in a non-limiting example, is very useful and effective in achieving the desired analysis goal. The balance may include quantitative measurements and/or qualitative measurements in conjunction with quantitative measurements, or imaging in conjunction with quantitative measurements. The methods and systems shown herein provide different fluorescent channels, where each channel may contain one or more specific molecular markers for specificity (e.g., quantitative information). The methods and systems shown herein may include or may be used with a microscope, examples of which may have the ability to observe and measure background from staining within cells (e.g., whether it is located within the cytoplasm, accumulated on a surface, within the nucleus, or in some other location), may be linked to images and/or qualitative information that has been generated for quantitative measurements. In this mode, linking to the original image from which the quantitative analysis result was generated provides the possibility for further analysis if the quantitative measurement results triggered an alarm or met a threshold indicating that further analysis is required. The examples herein may acquire background image data and information that produces staining within a cell in a sample within an analysis region 608. Such images and information allow a determination of whether the stain is produced intracellularly, for example, within the cytoplasm, within the nucleus, within the membrane of the cell, or within other organelles or cellular locations.

In some examples of the methods and systems shown herein, the scattering properties of the cell association quantification, the shape of the cell, and/or the size of the cell may be observed and measured, and may be used to confirm and/or delineate a sample. In some examples of the methods and systems shown herein, physical, optical, and biological/biochemical characteristics of a sample or portion of a sample may be observed and measured at the same time in the same device. All of these measurements and observations can be combined with a programmed processor or other processing system linked to various types of information for testing purposes (e.g., for a clinical test).

While conventional devices may be suitable for one or the other type of observation or measurement, they are not suitable for both epi-illumination and transmission illumination from the same light source; and there is no link between such different types of information. For example, in some examples shown herein, image information is available that yields quantitative measurements, and the systems and methods may be used for histomorphometry. Alternatively, the system can be applied to Pap smears, much like traditional cytology. It can be extended to any examination done using conventional microscopy techniques. In urine, at least some of the present examples allow the observation and analysis of crystals, not just cells. We can observe inorganic salt and chemical crystallization from urine samples that can produce certain quantitative readings in a portion of the graph. In addition to this, we can observe and analyze the cells and particles present in the blood, including the analysis of different types and populations of blood cells, such as but not limited to what might be seen in FIG. 1A, where the data for different regions are delineated. Image information for certain data regions may be acquired for further analysis of potential cellular images used to plot graphical or graphical measurements.

Some examples herein may combine the imaging configuration with the pathology configuration. For example, tissue preparation may be performed in a device or system designed to contain the optical elements shown herein (a system may be, or may include, for example, one or more modules for performing optical and other analyses on a sample), and such prepared material may be imaged in the platform. The image or analysis may then be sent to a server for image analysis, diagnosis, or performing digital pathology to effectively assist a pathologist in analyzing a sample.

The example methods, systems and devices shown herein, including, for example, the systems and devices shown in FIGS. 8C and 8D, provide a wide range of cell counting capabilities that may be applied together for analysis of a sample. Such cytometric capabilities include cytometric imaging, e.g., typically confined within a microscope; such microscopic imaging and imaging analysis of biological samples is provided by the devices and systems shown herein. In addition, the devices and systems shown herein are designed to provide spectrophotometric analysis of biological samples. Such image analysis includes dark field, bright field and other image analysis. There is shown a new and improved method of applying both epi-illumination and transmission illumination from the same light source, which allows us to more sensitively and accurately image blood samples. Separate measurements for RBCs, WBCs, and cells of these subclasses may be obtained for use with the methods shown herein. The images and spectrophotometric analysis shown herein may be used to identify and quantify different subpopulations of WBCs, facilitating the characterization of a blood sample and diagnosis of many clinical conditions. The devices and systems shown herein may be used to provide clinical reports including general chemical analysis information, nucleic acid-based analysis information, antibody (or protein, or epitope) -based analysis information, and spectrophotometric analysis information, as well as images of cells and samples being analyzed. The ability to generate such information and provide such reports, including images and other clinical information, is believed to provide novel and unexpected functions and results.

In addition, such information and reports may be generated in a short time (e.g., less than an hour, or less than 50 minutes, or less than 40 minutes, or less than 30 minutes, or other shorter time). In addition, such information and these reports may be generated from small volume samples, such as small volume blood or urine samples. The size of such small volume samples may be no more than about 500 μ L, or less than about 250 μ L, or less than about 150 μ L, or less than about 100 μ L, or less than about 75 μ L, or less than about 50 μ L, or less than about 40 μ L, or less than about 20 μ L, or less than about 10 μ L, or other small volumes. In some instances, where a sample is a blood sample, such small volume samples may be collected from a single fingertip blood draw. Typically, only one small volume of blood sample can be collected (e.g., the volume of blood may be about 250 μ L or less, or about 200 μ L or less, or about 150 μ L or less, or about 100 μ L or less, or about 50 μ L or less, or about 25 μ L or less, or other small volume).

As shown herein, clinical reports including cytometric information and images (including images, scatter plots, and other optical and imaging information), as well as general chemical analysis information, nucleic acid-based analysis information, antibody (or protein, or epitope) -based analysis information, and spectrophotometric analysis information, are believed to provide a broad and rich range of clinical information, aid in the diagnosis and description of many clinical situations, and provide technical advantages. Such reports may be prepared quickly at a point-of-care (point-of-care) station and may be communicated quickly (e.g., via wireless electronic transmission, landline, fiber optic, or other communication link) to a pathologist or other clinical professional for analysis and interpretation. Such expert analysis and interpretation, in turn, may be quickly relayed (e.g., via wireless electronic transmission, landline, fiber optic, or other communication link) back to the clinician and/or point-of-care (point-of-care) station taking care of the patient as quick feedback. Such rapid feedback, based on the information provided by the sample and the results of the analysis, can provide timely processing, if desired, or prevent unnecessary processing; the sample may be obtained, analyzed, or both at a point-of-care service or medical station. Such rapid analysis, reporting and feedback provides further benefits over time-consuming methods; but also potentially provides more efficient, and less expensive clinical services and treatments by allowing timely treatments and preventing unnecessary treatments. Such time consuming methods that may be avoided by the apparatus, systems, and methods shown herein include, but are not limited to: delays and inconvenience due to the need for the patient to go to a laboratory or clinic that is remote from the patient's home and the clinician authorized to take care of the patient; delays and possible sample degradation due to the transport of a sample from a collection site to a sample analysis site; delays due to the transmission of such analysis results to a medical doctor or other expert; delays in communicating an expert's view to the patient's clinician; the clinician is then given the opinion of an expert and the delay in the diagnosis and treatment process of the patient after the clinician. By using the methods, devices, and systems shown herein, these delays, invariance, and possible sample degradation may be reduced or eliminated.

Fig. 6A, 6B, 7, 8A, 8B, 8C, 8D and other schematic diagrams, as well as the examples of systems and devices illustrated herein, may provide functionality for cell counting, including use with one or more other sample analysis functions in a compact format. The invention applications for the novel devices and systems shown herein include novel cell counting functions and other sample analysis functions in the devices and systems shown herein. For example, the invention application of the devices and systems shown herein provides for the novel cell counting functionality shown herein to be used in conjunction with devices and systems for sample analysis via a common chemical unit; in conjunction with apparatus and systems for sample analysis by a nucleic acid analysis unit; in conjunction with devices and systems for sample analysis by antibody detection (e.g., ELISA) units; or a combination of several analytical units. A sample processing device as shown here may be designed to perform multiple tests on a sample. Such a sample may be a small volume sample.

In some examples, all sample detection operations and steps are performed on a single sample. In some instances, all sample testing operations and steps are performed by a single device or system, and may be performed within a single device packaged. Such systems and devices, including cytometers, particularly cytometers that provide image analysis and spectrophotometric or other optical analysis in a single unit, are believed to be novel and unexpected. Providing a system and apparatus comprising a cytometer, particularly a cell count that provides image analysis and spectrophotometer or other optical analysis in a single unit, is believed to provide advantages not possible with previous techniques.

Fig. 6A, 6B, 7, 8A, 8B, 8C, 8D and other schematic diagrams and examples of systems and devices shown herein provide cell counting functionality in a mobile form, and such devices and systems may be packaged in a sufficiently small enclosure to facilitate transport from one location to another. For example, such devices and systems may be used at a point-of-care (e.g., a doctor's office, a clinic, a hospital, a clinical laboratory, or other location) for ready delivery. For example, the apparatus and system may be used at a point-of-care service site (including, in addition to the point-of-care medical site described above, such as a pharmacy, a supermarket, or other retail or service site) for ready transmission. A point-of-care service site may include, for example, any location where a patient may receive a service (e.g., testing, monitoring, processing, diagnosis, guidance, sample collection, identity verification, medical services, non-medical services, etc.). Fixed-point service locations include, but are not limited to, a subject's home, a subject's work location, a medical service provider's location (e.g., doctor), hospital, emergency room, operating room, clinic, medical service professional's office, laboratory, retail store [ e.g., pharmacy (e.g., retail pharmacy, clinical pharmacy, hospital pharmacy), pharmacy, supermarket, food store, etc. ], transportation vehicles (e.g., cars, boats, trucks, buses, airplanes, motorcycles, ambulances, mobile units, fire trucks, emergency vehicles, law enforcement vehicles, police vehicles, or other vehicles used to transport a subject from one location to another, etc.), travel medical service units, mobile units, schools, nursery centers, screening locations, competition sites, medical assisted living homes, government offices, police, etc, An office building, tent, body fluid sample acquisition site (e.g., a blood collection center), entrance at or near a site that a subject may wish to enter, site at or near a device that a subject may wish to use (e.g., a computer if the subject wishes to use the computer), site where a sample processing device receives a sample, or any other point-of-service site described elsewhere herein.

Esoteric cell counting technique and specific cell counting markers

Many conventional high-grade or esoteric cytometric assays require a conventional system to measure a large number of markers on cells; typically these markers are measured simultaneously. Common detection methods in this field have been closely associated with high-function instruments, including, for example, 6 or more laser light sources and 18 different PMT tubes to measure all of these markers simultaneously. However, in many clinical situations, it is not necessary to measure multiple markers simultaneously. For example, in many clinical needs, one question of interest is to see how many cells are positively expressed for a marker at a glance; or how many cells are positively expressed for two or three combined markers; or expression of several other similar combination markers. Some examples herein provide a multiple marker co-staining scheme, one of which may contain, for example, a set of 10 markers, one of which may be a combination of a set of 3-4 or 5-6 markers; it is even possible to combine two markers with the same color. Some examples in the present system may also decrypt the complex image and information to determine which signal originated from which marker. This allows some instances of the present system to reduce their hardware requirements, i.e., the number of light sources, the number of channels used for sample analysis, and to perform other simplifications and synergies. Thus, complex cytometric assays can be very useful using a subset number of markers, or using non-simultaneous methods of applying and measuring markers in a predetermined pair combination. For example, some markers may be considered "gated" markers; such a marker is measured first, and if such an initial measurement is negative (e.g., the marker is not expressed, or is expressed only in small amounts, in one sample), then no further subsequent markers may need to be measured. In an example, such non-simultaneous methods and systems may reduce the sample volume required for analysis and may reduce the number of markers required for analysis (e.g., a subsequent marker is typically used in only a small portion of the sample being analyzed).

It will be appreciated that the use of imaging for cytometric analysis of a sample, such as a blood sample or urine sample, enables us to obtain a true cell count and may be more accurate than conventional cytometric methods that do not include such measurement. Imaging of a sample, including imaging of cells (or particles, or structures) within a sample, may indeed be more accurate than other methods, such as conventional cell counting methods. For example, conventional flow cytometry gating does not allow for true counts. Gating in flow cytometry is subjective and thus can vary greatly from system to system. Moreover, conventional flow cytometry methods do not provide an image of cells within a sample.

Some examples herein may also perform gating, but the gating is algorithmically based on different factors, including but not limited to the health of the patient. The classification method is revised by patient population to know whether they are healthy or diseased. Some examples herein may mark an abnormal patient and use for review. The self-learned gating may determine whether different gating is needed based on information conveyed about the patient's health. Thus, the gating of the sample in some of the examples shown herein may be accomplished by a programmed processor through computational rules, and the gating may be altered based on the patient's health.

In the example of a method and system for imaging, we may wish to minimize the amount and complexity of hardware required, and we may wish to reuse some or all of the sample as much as possible to reduce the amount of sample that needs to be acquired. Thus, the more functions we can capture from an image of a sample, the better we can obtain the maximum information from a sample, and possibly from a smaller sample size. Thus, the more information we obtain from a minimal number of images that distinguishes between different cell types, the more likely we will be to reduce the volume of sample that needs to be acquired.

Alternatively, in one non-limiting example, the cuvette for use in the microscope stage may be designed in the following form (see examples and elements shown in fig. 7, 8A and 8B). An intermediate duct layer comprises a very thin plastic film core 800 with pressure sensitive adhesive (psa) on both sides. One side is adhered to the light transmitting layer 606 and the other side is adhered to the top layer shaping the cover portion 612. The core is an extruded film, which is black in color, mainly for optical reasons such as prevention of light scattering and prevention of optical interference between different liquid conduits. The core film preferably has a uniform thickness along its length and width, and may be made of a film such as a black PET or black HDPE (polyethylene) extrusion. The psa sublayers on both sides tend to be as thin as possible to keep their entire fluid conduit tight and consistent (e.g., analysis region 608); but it needs to be thick enough to provide a good liquid seal around the liquid pipe. In an example, the psa adhesive that contributes to such a sample holder is substantially acrylic plastic, which has a high adhesion strength to low surface energy plastics. The liquid conduits, openings and other line-like structures on the middle layer face thereof may be made using laser cutting or die cutting operations.

This example also shows that magnetic elements, such as but not limited to magnetic spheres or disks, or metal spheres or disks that may be attracted by a magnet, may be incorporated into the cuvette. For example, the magnetic element may be included in or constitute a sample rack or cuvette shaping top layer. Magnetic elements may be used to simplify the hardware used to transport the cuvette. For example, the processing system may engage with magnetic structures within the cuvette to transport it without having to have an additional sample processing device.

While the invention has been described and illustrated with reference to certain specific examples, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additional steps and procedures may be made without departing from the spirit and scope of the invention. For example, non-stop materials may be used to create different reflective surfaces within the cuvette, or other surfaces that exist along a light path within the optical system. Optionally, the reflective surface is selected such that the reflection is only diffuse. Alternatively, the reflective surface is selected such that the reflection is only specular. Some examples may use a flat-top illumination design as specified by Coumans, f.a.w., van der Pol, e., & Terstappen, l.w.m.m. (2012), which appears in epi-fluorescence microscopy through a double microlens array. Cell counter, 81A: 324-331. doi:10.1002/cyto.a.22029, which is incorporated herein by reference in its entirety for all purposes.

Additionally, concentrations, amounts, and other numerical data may be displayed in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus the results should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a size range of about 1nm to 20nm should be interpreted to include not only the explicitly recited limits of 1nm and 20nm, but also to include individual sizes such as 2nm, 3nm, 4nm, and sub-ranges such as 10nm to 50nm, 20nm to 100nm, as well as other ranges.

Publications discussed or referenced herein are provided solely for their discovery prior to the filing date of the present application. This statement is not to be taken as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may differ from the actual publication dates which may need to be independently confirmed. All publications mentioned herein are incorporated herein by reference to show and describe the structures and/or methods in connection with which the publications are cited. The following applications are also incorporated herein by reference for various purposes. U.S. patent nos. 7,888,125; U.S. patent nos. 8,007,999; U.S. Pat. nos. 8,088,593; U.S. Pat. nos. 8,088,593; us patent 8,380,541; U.S. patent publication No. US 20120309636; PCT application numbers PCT/US 2012/057155; PCT application numbers PCT/US 2011/53188; PCT application No. PCT/US 11/53189; U.S. patent application serial No. 13/769,779; U.S. patent application 13/244,946; U.S. patent application 13/244,947; U.S. patent application 13/244,949; U.S. patent application 13/244,950; U.S. patent application 13/244,951; U.S. patent application 13/244,952; U.S. patent application 13/244,953; U.S. patent application 13/244,954; U.S. patent application 13/244,956; U.S. patent application 13/769,798; U.S. patent application 13/769,820; U.S. patent application 61/766,113; U.S. patent application No. 61/673,245; U.S. patent application 61/786,351; U.S. patent application 61/697,797; and U.S. patent application 61/733,886, which are hereby incorporated by reference in their entirety for all purposes.

This application claims priority to U.S. patent application serial No. 61/675,811, filing date 2012, 7/25; U.S. patent application serial No. 61/676,178, filing date 2012, 7 months and 26 days; us application 61/676,116, filing date 2013, month 2, day 18; and U.S. patent application 61/802,194, filed 2013, 3, 15, the inventions of which are incorporated herein by reference in their entirety for all purposes.

Contained herein is material subject to copyright protection. The copyright owner, the applicant herein, has no objection to the facsimile reproduction by anyone of the patent material and the invention, as it appears in the U.S. patent and trademark office patent files or records, but otherwise reserves all copyright rights whatsoever. The following remarks will apply: copyright 2012 and 2013, Theranos corporation.

While the preferred embodiment of the invention has been described in detail, it is possible to use various alternatives, modifications, and equivalents. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. Any structure, whether or not it is desirable, may be combined with any other structure, whether or not it is desirable. The appended claims should not be construed as including a meaning-plus-function limitation unless an analogous limitation has been expressly set forth in the claim using the phrase "means". It should be understood that, as used in this specification and throughout the claims that follow, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. It should also be understood that, as used in this description and throughout the claims that follow, the meaning of "in. Finally, as used in this specification and throughout the claims that follow, the terms "and" or "are intended to encompass both conjunctive and disjunctive meanings and may be used interchangeably unless the context clearly dictates otherwise. Thus, when the context uses "and" or "unless the context clearly dictates otherwise, the usage of the conjunction does not exclude the meaning of" and/or "(and/or)".

Claims (58)

1. A system for analyzing a sample, the system comprising:

a sample holder comprising a sample chamber for receiving said sample, at least a portion of said sample holder comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface;

an illumination source providing light that impinges on and passes through said optically transmissive surface;

wherein said sample holder is designed with the effect that said light from said illumination source simultaneously provides both epi-illumination and transmission illumination to a sample in the sample holder, wherein the light constituting the epi-illumination is directed from said illumination source onto said sample without having to be reflected at a surface of the optically transmissive material of the sample holder; light rays constituting the transmitted illumination travel within the optically transmissive material and reach the sample after at least one reflection from at least one surface of the optically transmissive material.

2. The system of claim 1, wherein the sample holder comprises a cuvette having an elongated conduit for receiving a sample.

3. The system of claim 1 or 2, wherein the sample holder contains one or more optically non-transmissive surfaces.

4. The system of claim 1 or 2, wherein at least a portion of the transmitted illumination is provided by totally internally reflected light from one surface.

5. The system of claim 2, wherein at least a portion of the transmitted illumination is provided by total internal reflection light within the cuvette.

6. The system of claim 1, wherein the sample holder contains two or more sample chambers for receiving samples.

7. The system of claim 2, wherein the cuvette has a rectangular horizontal cross-section.

8. The system of claim 2, wherein the cuvette has a circular horizontal cross-section.

9. The system of claim 2, wherein the cuvette has a vertical cross-section that is serrated.

10. The system of claim 2, wherein the cuvette has a stepped vertical cross-section.

11. The system of claim 1, wherein the sample holder is movable relative to the illumination source and to a plurality of positions, wherein the optically transmissive surface of the sample holder is illuminated by the illumination source at each of the positions.

12. The system of claim 1, wherein the illumination source comprises an annular light.

13. The system of claim 12, wherein the ring light is selected from a Light Emitting Diode (LED) based ring light and a laser based ring light.

14. The system of claim 1, further comprising a support structure comprising an optically transmissive surface and shaped to engage an optically transmissive surface of the sample holder.

15. The system of claim 1, further comprising a pressurizing device designed to hold the sample holder in a desired position, illuminated by the illumination source.

16. The system of claim 1, further comprising a detector configured to image at least a portion of a tube within the sample holder.

17. The system as claimed in claim 16, wherein the sample holder contains an elongated conduit designed to receive at least a portion of the sample, and wherein the detector is designed to image the entire elongated conduit within the sample holder.

18. The system of claim 16, wherein the sample holder is designed to hold the sample in a stable, non-flowing state during imaging.

19. The system of claim 16, wherein the sample holder is designed to hold a portion of the sample in a stable, non-flowing state and another portion in a flowing state during imaging.

20. The system of claim 16, wherein the illumination source is movable relative to the sample holder.

21. The system of any preceding claim, wherein the sample holder is designed to hold the sample in a flow state during imaging.

22. The system of claim 16, wherein said sample holder further comprises a liquid circuit completely confined within the sample holder, wherein the sample is located within said liquid circuit, the effect of which is to keep the sample separate from said detector.

23. The system of claim 22, wherein the sample holder is movable relative to the detector.

24. The system of claim 22, wherein the detector is movable relative to the sample holder.

25. The system of claim 1, wherein the sample holder and the illumination source comprise at least a portion of an optical analysis unit, and the system further comprises a clinical analysis unit for performing clinical analysis on the sample.

26. The system of claim 25, wherein the system is configured to provide each of the optical analysis unit and the clinical analysis unit with a aliquot of a sample from a single sample, the validity of which is such that the clinical analysis unit and the optical analysis unit can perform both optical analysis and clinical analysis on a sample portion at the same time.

27. The system of claim 25, wherein the clinical assay is selected from the group consisting of general chemical assays, nucleic acid assays, and enzyme-linked binding assays.

28. The system of claim 25, comprising a plurality of clinical analysis units, wherein each clinical analysis unit of the plurality of clinical analysis units is configured to provide a clinical analysis selected from the group consisting of general chemical analysis, nucleic acid analysis, and enzyme-linked binding analysis.

29. A cuvette having a sample chamber for receiving a sample; at least one portion of the cuvette comprises an optically transmissive material; the optically transmissive material has an optically transmissive surface and a reflective surface; wherein said optically transmissive surface and said optically reflective surface are operatively configured such that light transmitted through the optically transmissive surface simultaneously provides both epi-illumination and transmission illumination of said sample in the sample chamber, wherein light constituting the epi-illumination is directed from said illumination source to said sample without being reflected from a surface of the optically transmissive material of the sample holder; while the light rays constituting the transmitted illumination travel within the optically transmissive material and reach the sample after at least one reflection from at least one surface of said optically transmissive material.

30. The cuvette of claim 29, wherein the sample chamber contains an elongated tube.

31. The cuvette of claim 29, further comprising one or more optically non-transmissive surfaces.

32. The cuvette of claim 29, wherein at least a portion of the transmitted illumination is provided by partially internally reflecting light from a surface.

33. The cuvette of claim 29, wherein at least a portion of the transmitted illumination is provided by total internal reflection light from a surface.

34. The cuvette of claim 29, wherein the sample holder contains two or more sample chambers for holding samples.

35. The cuvette of claim 29, comprising a cross-sectional shape selected from a rectangular horizontal cross-sectional shape and a circular horizontal cross-sectional shape.

36. The cuvette of claim 29, comprising a cross-sectional shape selected from a sawtooth-like vertical cross-sectional shape and a staircase-like vertical cross-sectional shape.

37. A cuvette comprising a sample chamber having an optically transmissive bottom, said cuvette having an outer surface comprising at least one convex or one concave structure for providing mechanical support to the cuvette.

38. The cuvette of claim 37, wherein the at least one convex or concave structure comprises a cross-sectional shape selected from the group consisting of rectangular, triangular, circular, and semi-circular.

39. The cuvette of claim 37, wherein the at least one convex or concave surface structure is configured to provide a pathway for internally reflected light within the cuvette.

40. The cuvette of claim 37, wherein the at least one convex or concave structure comprises a surface, wherein the surface is configured to reflect light within the cuvette.

41. A method for distinguishing a cell from a sample containing a plurality of cells, comprising:

(a) placing said sample in a sample holder having a sample chamber for receiving the sample; at least one portion of the sample holder contains an optically transmissive material; the optically transmissive material has an optically transmissive surface and a reflective surface; wherein the optically transmissive surface and the optically reflective surface are operatively configured such that light transmitted through the optically transmissive surface simultaneously provides both epi-illumination and transmission illumination to the sample in the sample chamber, wherein light constituting the epi-illumination is directed from the illumination source to the sample without being reflected at a surface of the optically transmissive material of the sample holder; while the light rays constituting the transmitted illumination travel within the optically transmissive material and reach the sample after at least one reflection from at least one surface of said optically transmissive material;

(b) Illuminating the sample holder may be effective to provide both epi-illumination and transmission illumination of the sample;

(c) identifying a cell within the sample.

42. The method of claim 41, wherein said validating comprises discriminating said cell with a probe configured to image at least a portion of said sample chamber.

43. The method of claim 43, wherein the sample chamber contains an elongated tube.

44. A method of measuring a component of interest in cells of a population of cells in a sample, comprising:

a) obtaining a quantitative measurement of a marker expressed in cells of the population of cells within the sample;

b) determining, with the aid of a computer, an approximate number of cells of the population in the sample based on the measurements of part a);

c) adding to the sample an amount of a cellular marker, wherein the amount of said cellular marker added is based on the results obtained in part b), and wherein the cellular marker specifically binds to the component of interest in the cells of the population and is readily detectable;

d) detecting cells in the sample by the label bound to the component of interest;

e) Determining the amount of the component of interest in the cells of the population of cells in the sample based on the amount of the label bound to the component of interest.

45. The method of claim 44, wherein the sample holder comprises a sample holder selected from the sample holder of claim 29 and the sample holder of claim 37.

46. A method of focusing a microscope, comprising:

a) mixing a sample containing a target to be microscopically analyzed with a reference particle of known size effective to produce a mixture containing the sample and the reference particle;

b) placing the mixture of step a) in the optical path of a microscope;

c) exposing the mixture of step a) to a light wave designed to observe the reference particle;

d) focusing the microscope according to the position of the reference particle in the mixture or according to the sharpness of the image of the reference particle.

47. The method of claim 46, wherein the mixture comprising the sample and a reference particle is placed in a sample holder selected from the sample holder of claim 29 and the sample holder of claim 37.

48. A method for distinguishing a cell from a sample containing a plurality of cells, comprising:

(a) Analyzing a cell of the plurality of cells for at least one of: (i) expression of cell surface antigens; (ii) the amount of one cell surface antigen; (iii) cell size;

(b) detecting the cells of (a) for at least one purpose: (i) the size of the cell nucleus; or (ii) nuclear shape;

(c) detecting the cells of (a) and (b), and quantitatively analyzing the light scattering of the cells,

wherein the combined information from steps (a), (b), and (c) is used to identify the cell in a sample containing a plurality of cells.

49. The method of claim 48, wherein the plurality of cells are disposed in a sample holder selected from the sample holder of claim 29 and the sample holder of claim 37.

50. A system for imaging a sample, comprising:

a sample rack is arranged on the upper portion of the frame,

a light source for illuminating an object placed in the sample holder,

an objective lens designed to collect and focus light scattered by a target in the sample holder, wherein the scattered light comprises light scattered at a plurality of scattering angles,

an optical aperture passes light from the objective lens,

an additional lens for focusing light from said objective lens onto said optical aperture, wherein said optical aperture is designed to allow only a portion of light focused by said objective lens to pass through, and said portion of light allowed to pass through said aperture is comprised of scattered light at only a portion of said plurality of scattering angles.

51. A system for imaging a sample, the system comprising:

a sample container containing said sample, wherein said sample container,

a stage comprising a sample vessel having an optically transparent surface,

a light source for illuminating the presence of a species in the sample through the stage,

wherein the sample container has an interface surface for engaging an optically transparent surface of the sample container receiver, the interface surface conforming to the optically transparent surface without significant distortion when light passes through the interface surface.

52. The system of claim 51, wherein the interface surface of the sample container is formed of a high molecular polymer material.

53. The system of claim 51, wherein the sample vessel interface surface is formed of a material that is more flexible than a material used for the optically transparent surface of the sample vessel receiver.

54. The system of claim 51, further comprising a pressurizing unit for pressurizing the interface surface to conform to a shape of the sample container receiver optically transparent surface.

55. The system of claim 51, further comprising a processing unit configured to couple with the sample container, facilitate transporting the sample container up and down the stage, and increase mechanical strength of the sample container.

56. The system of claim 51, further comprising an opaque processing unit configured to couple to the sample container.

57. A system as in claim 51, wherein all imaging of the sample is possible without passing substantially straight light through one surface and out the opposite surface to a detector.

58. The system of claim 51, wherein the light source does not transmit light to a detector on an opposite side of the sample container than the one on the opposite side of the sample container.