HK1154653B - Compensator for multiple surface imaging - Google Patents

Description

Compensator for multi-surface imaging

Technical Field

The present invention relates generally to the field of imaging and evaluating analytical samples. More particularly, the present invention relates to techniques for imaging and evaluating analytical samples on multiple surfaces of a support structure using compensators.

Background

There are more and more applications for imaging an analytical sample on a support structure. These support structures may include a plurality of substrates on which the biological samples are placed. For example, these substrates may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) probes that are specific for nucleotide sequences present in genes of humans and other organisms. Various DNA or RNA probes may be attached to specific locations in a small geometric grid or array on such a support structure. Depending on the technique employed, the sample may be affixed to the support structure at random, semi-random, or predetermined locations. A test sample, e.g., from a known human or organism, can be exposed to the array or grid such that complementary genes or fragments hybridize to probes at various sites on the substrate surface. In some applications (e.g., sequencing), a template or fragment of genetic material may be placed at various sites and nucleosides or other molecules allowed to hybridize to the template to determine the identity or sequence of the template. Thus, these sites can be examined by the following procedure: light of a particular frequency is scanned over these sites, identifying which genes or fragments in the sample were present using the fluorescence at those sites where the genes or fragments hybridise.

These substrates are often referred to as microarrays, gene or genomic chips, DNA chips, gene arrays, and the like, and can also be used for expression profiling, monitoring expression levels, genotyping, sequencing, and the like. For example, diagnostic applications may include: the genetic makeup of a particular patient is evaluated to determine whether a disease state or a sign of a particular condition exists. The reading and evaluation of such substrates is an important part of the present invention. Although microarrays allow presentation of separate biological components for batch processing and individual detection, the number of components that can be detected in a single experiment is limited by the resolution of the system. In addition, bulk reagents used in some processes are expensive and it is desirable to reduce the amount used. The present invention provides methods and compositions that can increase the efficiency of array-based assays to counteract these limitations. Other advantages are provided and will be apparent from the following description.

Disclosure of Invention

The present invention provides a new method for imaging and evaluation of analytical samples to extend the application of imaging and evaluation subsystems to multiple surfaces supporting the samples. The support structure may be, for example, a flow cell, allowing reagents to flow through the flow cell and react with the biological sample. The excitation radiation generated by the at least one radiation source may be used to excite the biological sample on a plurality of surfaces. In this way, fluorescent radiation may be emitted from the biological sample and subsequently captured and detected using detection optics and on at least one detector. The returned radiation may then be used to generate image data. Such imaging of multiple surfaces may be done sequentially or simultaneously. In addition, the techniques of the present invention may be used in any of a variety of imaging systems. For example, the present techniques facilitate both microfluorescent and total internal emission (TIR) approaches. In addition, the imaged biological sample may be presented in random locations or patterns on the surface of the support structure.

Accordingly, the present invention provides a method of imaging a biological sample, the method comprising detecting radiation emitted by a first emissive component of the biological sample disposed on a first surface of a flow cell using a detector. The flow units are positioned at an imaging station. The method also includes inserting corrective optics between the detector and the flow cell. The method also includes detecting radiation emitted by a second emissive component of the biological sample disposed on a second surface of the flow cell using the detector and the corrective optics. The first and second surfaces are both arranged in a manner whereby one of the surfaces is placed between the detector and the other surface. In addition, the corrective optics reduce the detected aberrations caused by this arrangement at one of the surfaces. The steps of the method are repeated while the flow cell remains at the imaging station.

The invention also provides an imaging system for detecting radiation on a multi-surface flow cell. The imaging system includes a multi-surface flow cell having first and second emissive components of a biological sample disposed on first and second surfaces of the flow cell. The imaging system also includes an optical train including an objective lens, imaging optics configured to focus the optical train on the first emission component through the objective lens, and correction optics configured to focus the optical train on the second emission component and configured to reduce a detected aberration on the first or second emission components. The imaging system further includes a radiation source configured to direct excitation radiation to the first and second emissive components. In addition, the imaging system includes detection optics configured to capture emission radiation returned from the first and second emission components by the optical train. Furthermore, the imaging system comprises a detector for detecting the captured radiation.

Drawings

FIG. 1 is a view of an imaging system for biological samples according to the present invention;

FIG. 2 is a perspective view of an exemplary line of radiation directly illuminating a semi-confocal radiation biological site from a support structure surface and semi-confocal return radiation to a detector in accordance with the present invention;

FIG. 3 is a cross-sectional view of an exemplary support structure having excitation radiation directed at both surfaces of the support structure in accordance with the present invention;

FIG. 4 is a perspective view of an exemplary support structure having an array of spatially arranged patterned biological component sites in accordance with the present invention;

FIG. 5 is a perspective view of an exemplary support structure having a random spatial distribution of biological component sites according to the present invention;

FIG. 6 is a cross-sectional view of an exemplary structure having excitation radiation directed at multiple surfaces of a support structure in accordance with the present invention;

FIG. 7 is a diagram illustrating exemplary dimensions between an objective lens and a support structure according to the present invention;

FIG. 8 is an exemplary plot of spherical aberration versus upper substrate thickness for the support structure of FIG. 7 in accordance with the present invention;

FIG. 9A illustrates an exemplary image expected of first and second surfaces of a support structure obtained with an upper surface thickness of 300 microns (100 microns plus fluid) without correction optics, wherein the second surface is optimized by an imaging system;

FIG. 9B illustrates an exemplary image expected of the first and second surfaces of the support structure obtained with an upper surface thickness of 340 microns (100 microns plus fluid) without correction optics;

FIG. 10A illustrates exemplary objective imaging of a second surface without compensator assistance in accordance with the present invention;

FIG. 10B illustrates exemplary objective imaging of a first surface with compensator assistance in accordance with the present invention;

FIG. 11 is an exemplary compensator design according to the present invention including a first objective lens and a second objective lens that may replace the first objective lens in the optical train;

FIG. 12 is another exemplary compensator design according to the present invention including a corrective device that can be inserted between the objective lens and the support structure;

FIG. 13 is another exemplary compensator design according to the present invention including a correction ring;

FIG. 14 is another exemplary compensator design according to the present invention, including an infinite space compensator;

FIG. 15 is a perspective view of an exemplary flow cell assembly using patterned adhesive to form channel features in accordance with the present invention;

FIG. 16 is a perspective view of another exemplary flow cell assembly using patterned adhesive to form channel features in accordance with the present disclosure;

FIG. 17 is a process flow diagram of an exemplary method of assembling a flow cell using patterned adhesive to form channel features in accordance with the present invention;

FIG. 18 is a view of a biological specimen imaging system employing one radiation source and two detectors and configured to sequentially scan multiple surfaces of a support structure in accordance with the present invention;

FIG. 19 is a view of a biological specimen imaging system employing two radiation sources and two detectors and configured to sequentially scan multiple surfaces of a support structure in accordance with the present invention;

FIG. 20 is a view of a biological sample imaging system employing two radiation sources and two detectors and a focusing lens along an excitation path and configured to synchronously scan multiple surfaces of a support structure in accordance with the present invention;

FIG. 21 is a view of a biological sample imaging system employing two radiation sources and two detectors and using focusing lenses along the excitation and emission paths and configured to synchronously scan multiple surfaces of a support structure in accordance with the present invention;

FIG. 22 is a view of a biological sample imaging system employing multiple radiation sources and multiple detectors and using focusing lenses along the excitation path and configured to synchronously scan multiple surfaces of a support structure in accordance with the present invention;

FIG. 23 is a view of a TIR biological sample imaging system according to the present invention;

FIG. 24 is a cross-sectional view of an exemplary support structure, prism and lens objective for TIR imaging of the bottom surface of a flow path according to the present invention;

FIG. 25 is a cross-sectional view of an exemplary support structure, prism and lens objective for TIR imaging of the top surface of a flow path according to the present invention;

FIG. 26 is a cross-sectional view of another exemplary support structure, prism and lens objective for use in TIR imaging of the top surface of the flow path according to the present invention; and

FIG. 27 is a cross-sectional view of an exemplary support structure heated at the top and bottom surfaces according to the present invention.

Detailed Description

Referring now to the drawings, and initially to FIG. 1, a biological specimen imaging system 10 is illustrated. The biological sample imaging system 10 is capable of imaging a plurality of biological components 12, 14 within a support structure 16. For example, in the illustrated embodiment, the first biological component 12 may be presented on a first surface of the support structure 16, while the second biological component 14 may be presented on a second surface of the support structure. The support structure 16 may be, for example, a flow cell having an array of biological components 12, 14 on its interior surfaces 18, 20, which are generally facing each other and through which reagents, irrigants and other liquids may be introduced for binding nucleosides or other molecules to the sites of the biological components 12, 14. Support structure 16 may be made in conjunction with the techniques of the present invention, or support structure 16 may be purchased or otherwise obtained from a separate manufacturer. The fluorescent label on the molecule of the binding member may comprise, for example, a dye that fluoresces upon excitation by suitable excitation radiation. The assay methods that include the use of fluorescent labels and that can be used in the device or the methods set forth herein include other methods set forth herein, such as genotyping assays, gene expression analysis, methylation analysis, or nucleic acid sequencing analysis.

Those skilled in the art will recognize that: the flow cells or other support structures may take any of a variety of arrays that are well known in the art in order to achieve similar results. Furthermore, known methods of forming arrays can also be used and, if appropriate, modified in accordance with the techniques set forth herein to create flow cells or support structures having multiple active surfaces in the detection methods set forth herein. Such an array may be formed by any known technique for depositing the biological components of the sample randomly or in a predetermined pattern on the surface of the support. In predetermined embodiments, the nucleic acid-expanded cluster array may be prepared using the methods discussed in U.S. Pat. No.7,115,400, U.S. patent application publication No.2005/0100900, PCT publication No. WO00/189657, or PCT publication No. WO98/44151, which are incorporated herein by reference. Such methods are known, as are bridge amplification or solid-phase amplification, and are particularly useful for sequencing applications.

Other exemplary random and structured methods for use in the present invention include, but are not limited to, combining beads with solid supports, for example, U.S. Pat. Nos. 6,355,431, 6,327,410 and US6,770,441, U.S. patent application publication Nos. 2004/0185483 and US 2002/0102578; and PCT publication No. wo00/63437, which are incorporated herein by reference. Beads can be placed at various discrete locations, for example, on the side walls of a solid support, and a single bead can be provided at each location.

The present invention may also be used with any other type of array and method of constructing such arrays known in the art. Commercial microarrays that can be used include:microarray or array according to what is sometimes called VLSIPS^TMOther microarrays synthesized by (Very Large Scale Immobilized Polymer Synthesis) technology, as described in U.S. Pat. Nos.5,324,633; 5,744,305; 5,451,683, respectively; 5,482,867, respectively; 5,491,074, respectively; 5,624,711, respectively; 5,795,716; 5,831,070, respectively; 5,856,101, respectively; 5,858,659; 5,874,219; 5,968,740; 5,974,164; 5,981,185, respectively; 5,981,956, respectively; 6,025,601, respectively; 6,033,860, respectively; 6,090,555; 6,136,269, respectively; 6,022,963; 6,083,697, respectively; 6,291,183, respectively; 6,309,831, respectively; 6,416,949, respectively; 6,428,752 and 6,482,591, which are hereby incorporated by reference. The methods of the invention may also be used with a spot microarray, an exemplary spot microarray being CodeLink from Amersham biosciences^TMAnd (4) array. Another microarray useful in the present invention is a microarray made using an ink jet printing process, for example, using the sureprint of Agilent Technologies^TMProvided is a technique.

The sites or features of the array are generally discrete and spatially separated from each other. The size of the sites and/or the spacing between the sites can be varied to enable the array to have a higher density, a medium density, or a lower density. High density arrays have the property that the spacing of sites is less than about 15 μm. Medium density arrays have the property of having site spacing of about 15 to 30 μm, while low density arrays have the property of having site spacing greater than 30 μm. Arrays useful in the present invention may have sites less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm apart. The device or method of the invention is capable of imaging an array with sufficient resolution to distinguish sites at the above-mentioned densities or density ranges.

As explained herein, the surfaces used in the devices or methods of the present invention are generally manufactured surfaces. It is also possible to use a natural surface or a surface of a natural support structure, however, the invention may also be implemented in embodiments where the surface is not a natural material or is not a surface of a natural support structure. Thus, the components of the biological sample can be removed from the natural environment and adhere to the manufacturing surface.

Any of a variety of biological components can be present on a surface for use in the present invention. Exemplary components include, but are not limited to, nucleic acids such as DNA or RNA, proteins such as enzymes or receptors, polypeptides of these natural components, nucleotide transferases, amino acids, saccharines, cofactors, metabolic factors or derived factors. Although the devices and methods of the present invention are described herein with reference to components of biological samples, it should be understood that other samples or components may be used with advantage. For example, synthetic samples can also be used, e.g., combinatorial libraries or libraries of components of known or suspected substances having a desired structure or function. Thus, the apparatus or method can be used to synthesize a variety of components and/or shielding components for a desired structure or function.

Returning to the exemplary system shown in FIG. 1, biological specimen imaging system 10 includes at least a first radiation source 22, but may also include a second radiation source 24 (or other sources). The radiation sources 22, 24 may be lasers having different wavelengths. The wavelength selection of the laser is generally dependent on the fluorescent properties of the dyes used to image the component sites, and the use of multiple different wavelength lasers may allow for discrimination of dyes at various sites in the support structure 16, and the imaging operation may be continued by successive acquisitions of some images to enable identification of molecules at these component sites using image processing and reading logic generally known in the art. Other radiation sources known in the art can also be used, including, for example, arc lamps or quartz halogen lamps. Particularly effective radiation sources are those capable of electromagnetic radiation in the Ultraviolet (UV) range (about 200 to 390nm), visible light (VIS) range (about 390 to 770nm), Infrared (IR) range (about 0.77 to 25 μm), or other electromagnetic spectrum.

For ease of description, detection methods such as those based on fluorescence may be used. However, it should be understood that other detection methods may be used in conjunction with the devices and methods set forth herein. For example, a variety of different emission types may be detected, e.g., fluorescence, luminescence, or chemiluminescence. Thus, the component to be detected may be labelled with a fluorescent, luminescent or chemiluminescent compound or moiety. Detection using signals other than optical signals may also be performed using apparatus and methods similar to the embodiments herein to improve detection to accommodate the particular physical characteristics of the detected signals.

The output of radiation sources 22, 24 may be directed through conditioning optics 26, 28 for filtering and shaping the beam. For example, in contemplated embodiments of the present invention, conditioning optics 26, 28 may produce a substantially linear beam of radiation and combine the beams of multiple lasers, as discussed in U.S. patent No.7,329,860. The laser module additionally comprises measuring means for recording the individual laser powers. The measurement of power can be used as a feedback mechanism to control the length of time that an image is recorded, thereby achieving a uniform exposure and a more appropriate signal.

After passing through conditioning optics 26, 28, the beam may be directed to directing optics 30 for redirecting the beam from radiation sources 22, 24 to focusing optics 32. The directing optics 30 may include dichroic mirrors configured to redirect the light beam to the focusing optics 32 while also allowing a wavelength of the backward light beam to pass therethrough. The focusing optics 32 may confocally direct the radiation toward one or more surfaces 18, 20 of the support structure 16 on which the respective biological components 12, 14 are located. For example, focusing optics 32 may include a microscope objective, confocally directing and focusing radiation sources 22, 24 along a line to surfaces 18, 20 of support structure 16.

The biological component sites on the support structure 16 may fluoresce at a wavelength in response to the excitation beam and thereby return radiation for imaging. For example, the fluorescent component may be produced by a method of fluorescently labeling nucleic acids, which may hybridize to the remaining molecules in the component or to a fluorescently labeled nucleotidyl transferase, thereby combining into oligo (poly) nucleotides using a polymerase. As mentioned above, the fluorescent properties of these components may be altered by incorporating agents into the support structure 16 (e.g., by cleaving the dye from the molecule, preventing adhesion of other molecules, adding quenching agents, adding energy transfer acceptors, etc.). As will be appreciated by those skilled in the art, the wavelength at which the sample dye is excited and the wavelength at which the fluorescence is excited will depend on the absorption and emission spectra of the particular dye. This returned radiation may propagate back through the guiding optics 30. Such a backward beam may generally be directed to directing optics 34 that serve to filter the beam to separate out the different wavelengths within the backward beam and direct the backward beam to at least one detector 36.

The detector 36 may be based on any suitable technology and may be, for example, a Charge Coupled Device (CCD) sensor for generating pixel-based image data based on photon transition positions in the device. However, it should be understood that any of a variety of other detectors may be used, including, but not limited to, a detector array configured for Time Delay Integration (TDI) operation, a Complementary Metal Oxide Semiconductor (CMOS) detector, an Avalanche Photodiode (APD) detector, a geiger-mode photon counter, or any other suitable detector. The TDI mode detector may be coupled using line scanning, as discussed in U.S. patent No.7,329,860.

The detector 36 may generate image data, for example, having a resolution between 0.1 and 50 μm, and then transmit to the control/processing system 38. In general, control/processing system 38 may perform various operations, such as analog-to-digital conversion, scaling, filtering, and correlation of data in multiple frames to properly and accurately image multiple sites at particular locations on a sample. Control/processing system 38 may store the image data and may ultimately send the image data to a post-processing system (not shown) where the data is analyzed. Depending on the type of sample, the reagents used and the processing performed, a series of different uses of the image data may be made. For example, the nucleotide sequence may be derived from image data, or the data may be used to determine the presence of a particular gene, characterize one or more molecules at the constituent sites, and so forth. The operation of the various components illustrated in fig. 1 may also be coordinated with the control/processing system 38. In particular applications, control/processing system 38 may include hardware, firmware, and software designed to control the operation of radiation sources 22, 24, the movement and focusing of focusing optics 32, the operation of translation system 40 and detection optics 34, and the acquisition and processing of signals from detector 36. Control/processing system 38 may thereby store the processed data and further process the data to produce reconstructed images of the illuminated sites fluorescing in support structure 16. The image data may be analyzed by the system itself or may be stored for analysis by other systems and at different times after imaging.

The support structure 16 may be supported on a translation system 40 to allow for focusing and movement of the support structure 16 after and during imaging. The stage may be configured to move the support structure 16 to change the relative positions of the radiation sources 22, 24 and detector 36 with respect to the surface to which the biological components are bonded to facilitate continuous scanning. The translation system 40 movement may be in one or more dimensions, including, for example, one or two dimensions, labeled as the X and Y dimensions, perpendicular to the direction of transmission of the excitation radiation. In particular embodiments, translation system 40 may be configured to move in a direction perpendicular to the scan axis of the detector array. The translation system 40 useful in the present invention may also be configured to move along the dimension in which the line of excitation radiation propagates, which is generally labeled as the Z dimension. Movement in the Z dimension also facilitates focusing.

Fig. 2 is a diagram illustrating an exemplary semi-confocal line scanning method of imaging support structure 16, in the illustrated embodiment support structure 16 includes upper substrate 42 and lower substrate 44, and an interior volume 46 between upper and lower substrates 42, 44. The upper and lower substrates 42, 44 may be made of any material, but are preferably made of a substrate material that is substantially transparent at the wavelengths of the excitation radiation and the fluorescent backward beam, thereby allowing the excitation radiation and the returning fluorescent emission to pass without significant loss of signal quality. For example, in the epi-fluorescence imaging arrangement shown, one surface through which the radiation passes is substantially transparent at the relevant wavelength, while the other surface (the surface through which the radiation does not pass) is less transparent, translucent, or even opaque or reflective. The upper and lower substrates 42, 44 may contain biological components 12, 14 on their respective inward facing surfaces 18, 20. As discussed above, the interior volume 46 may include, for example, one or more internal channels of a flow cell through which reagent fluid may flow.

The support structure 16 may be illuminated by excitation radiation 48 along a line of radiation 50. Radiation line 50 may be formed by excitation radiation emitted by radiation sources 22, 24 and directed by directing optics 30 through focusing optics 32. The radiation sources 22, 24 may produce beams of light that, after being processed and shaped, provide a linear cross-section or radiation that includes multiple wavelengths of radiation for producing fluorescence of wavelengths different from the corresponding wavelengths of the biological components 12, 14 depending on the particular dye used. The focusing optics 32 may then semi-confocally direct the excitation radiation 48 onto the first surface 18 of the support structure 16 for illuminating the site of the biological component 12 along the line of radiation 50. In addition, the support structure 16, the directing optics 30, the focusing optics 32, or some combination thereof may be slowly translated such that the generated radiation 50 continuously illuminates the above-mentioned components, as indicated by arrows 52. This translation creates a continuous scan area 54, allowing the entire first surface 18 of the support structure 16 to be progressively illuminated. As will be described in further detail below, the same process may also be used to progressively illuminate the second surface 20 of the support structure 16. In fact, the treatment method may be used for multiple surfaces in the support structure 16.

Exemplary methods and apparatus for linear scanning are described in U.S. patent No.7,329,860, incorporated herein by reference, which discusses a linear scanning apparatus having a detector array for achieving confocal characteristics of the scan axis by limiting the scan axis dimensions of the detector array. More particularly, the scanning device may be configured such that the detector array has a rectangular dimension such that the shorter dimension of the detector is in the direction of the scan axis dimension and the imaging optics may be arranged to direct a rectangular image of the sample area to the detector array such that the shorter dimension of the image is also in the direction of the scan axis dimension. In this way, a semi-confocal feature can be achieved because the confocal characteristic occurs on a single axis (i.e., the scan axis). Thus, the detection may have features specific to the substrate surface, thereby suppressing signals due to solvent surrounding the features. As discussed herein, the apparatus and method discussed in U.S. Pat. No.7,329,860 may be further modified so that two or more surfaces of the support may be scanned.

Detection devices and methods other than line scanning may also be used. For example, spot scanning may be used, such as discussed in U.S. patent No.5,646,411, which is incorporated herein by reference. Wide angle area scanning may be used with or without scanning monitoring. TIR methods may also be used, as will be further explained herein.

Generally, as illustrated in FIG. 2, the radiation 50 used to image the sites of the biological components 12, 14 according to the present invention may be a continuous or intermittent line. Thus, certain embodiments of the present invention may include broken lines such that most of the confocal or semi-confocal guided beam of radiation may still illuminate most of the points along the line of radiation 50. These interrupted beams may be generated by one or more sources that provide the excitation radiation 48 in a spot or scanning manner. These beams, as described above, may be directed confocally or semi-confocally to the first or second surfaces 18, 20 of the support structure 16 to illuminate the sites of the biological components 12, 14. Using the continuous semi-confocal line scanning method described above, the support structure 16, the directing optics 30, the focusing optics 32, and some combination thereof may be slowly advanced as indicated by arrow 52 to sequentially irradiate a scanning area 54 along the first or second surface 18, 20 of the support structure 16, thereby sequentially irradiating the site areas of the biological components 12, 14.

It should be noted that the system generally forms and directs both the excitation radiation and the return radiation simultaneously for imaging. In certain embodiments, confocal scanning may be used such that by scanning the excitation beam through the objective lens, the optical system directs the excitation point or spot across the biological component. The detection system images the emission formed by the excitation spot on the detector without scanning the backward beam. This is due to the fact that the backward beam is collected by the objective lens and separated from the excitation beam optical path before returning through the scanning device. The backward beam will therefore appear at a different point on the detector 36, depending on the field angle of the original excitation point in the objective lens. As the excitation point is scanned across the sample, an image of the excitation point appears in the shape of a line on the detector 36. This technique is useful if the scanning device for some reason is not able to collect the backward beam of the sample. Examples are holographic and acousto-optical scanning devices, which are capable of scanning a light beam at very high speeds and also of using diffraction to produce the scan. Thus, the scanning performance is a function of wavelength. The backward beam of emitted radiation is at a different wavelength than the excitation beam. Alternatively or additionally, the emission signal may be collected in sequence after a sequencing-by-sequencing excitation at a different wavelength.

In particular embodiments, the apparatus or method of the present invention may be at least about 0.01mm²The rate of/sec detects features on a surface. Faster rates may also be used, depending on the particular application of the invention, including,for example, the velocity is at least about 0.02mm in terms of the scan area or detection area²/sec，0.05mm²/sec，0.1mm²/sec，1mm²/sec，1.5mm²/sec，5mm²/sec，10mm²/sec，50mm²/sec，100mm²Sec or faster. If desired, for example, to reduce noise, the upper limit of the detection rate is about 0.05mm²/sec，0.1mm²/sec，1mm²/sec，1.5mm²/sec，5mm²/sec，10mm²/sec，50mm²Sec or 100mm²/sec。

In some embodiments, the support structure 16 may be used in a manner where it is desired to place the biological component on only one surface. However, in many embodiments, the biological material is placed on multiple surfaces of the support structure 16. For example, FIG. 3 illustrates a typical support structure 16 in which biological material is adhered to a first surface 18 and a second surface 20. In the illustrated embodiment, an adhesion layer 56 is formed on both the first surface 18 and the second surface 20 of the support structure 16. The first excitation radiation 58 source may be used to illuminate one of a number of sites of the biological component 12 on the first surface 18 of the support structure 16 and return a first fluorescent emission 60 from illuminating the biological component 12. Simultaneously or sequentially, a second source of excitation radiation 62 may be used to illuminate one of a number of sites of the biological component 14 on the second surface 20 of the support structure 16 and return a second fluorescent emission 64 from the illuminated biological component 14.

Although the embodiment illustrated in FIG. 3 shows that excitation from source 58 and source 62 may be from the same side of support structure 16, it should be understood that the optical system may be configured to be incident on surfaces on opposite sides of support structure 16. Taking fig. 3 as an example, upper surface 18 may be illuminated by excitation source 58 as shown, while lower surface 20 may be illuminated from below. Also, the emission may be detected from one or more sides of the support structure. In a particular embodiment, different sides of the support structure 16 may be activated by the same radiation source by first irradiating one side and then flipping the support structure so that the other side is brought into the proper position for the radiation source to activate.

The distribution of the biological components 12, 14 may follow many different patterns. For example, fig. 4 illustrates a support structure 16 in which biological components 12, 14 at sites or features on the first and second surfaces 18, 20 are uniformly distributed in a spatially arranged pattern 66 of biological component sites 68. For example, a type of microarray may be used in which the locations of the individual biocomponent sites 68 are in a regular spatial pattern. The pattern may include sites at predetermined locations. In contrast, in other types of biological imaging arrays, biological components adhere to the surface of sites that occur at random or statistically distributed varying locations, such that imaging of the microarray can be used to determine the location of each of the different biological component features. Thus, although the above-described feature patterns are the product of a synthesis or manufacturing process, they need not be predetermined.

For example, fig. 5 illustrates a support structure 16 in which sites on both the first and second surfaces 18, 20 are in a random spatial distribution 70 of biological component sites 72. However, with both a fixed array 66 and a random distribution 70 of biological sample sites, imaging of multiple surfaces 18, 20 of the support structure 16 is possible. In addition, it should be noted that in both examples, the biological components at each site may be composed of a population of identical molecules or may be composed of a random mixture of different molecules. Further, the density of the biological sample may vary and may be at least 1000 sites per square millimeter.

The techniques of the present invention provide varying physical arrangements of the multiple surfaces in the support structure 16 and varying deployment of sites within the composition on these surfaces. As discussed above with reference to fig. 2 and 3, in an embodiment, the support structure 16 has a first surface 18 and a second surface 20, and the first excitation radiation source 58 can irradiate sites of the biological component 12 on the first surface 18 and return first fluorescent radiation 60, while the second excitation radiation source 62 can irradiate sites of the biological component 14 on the second surface 20 and return a source of second fluorescent emissions 64, as shown in fig. 3. Thus, the components of the sample volume between the two surfaces need not be detected and can be suppressed. Selective detection of the support structure surface provides better detection of the support structure volume relative to adjacent surfaces and the surface relative to one or more surfaces of the support structure.

In more complex configurations, it is useful to illuminate more than two surfaces. For example, fig. 6 illustrates support structure 16 having N substrates, including first substrate 42, second substrate 44, N. The substrates define M surfaces including a first surface 18, a second surface 20, an M-3 surface 80, an M-2 surface 82, an M-1 surface 84, and an M surface 86. In the illustrated embodiment, not only the first surface 18 and the second surface 20 of the support structure 16 may be illuminated, but all M number of surfaces may be illuminated. For example, an excitation radiation source 88 may be used to irradiate a biological component site on the mth surface 86 of the support structure 16 and return fluorescent emissions 90 from the irradiated biological component. For support structures having multiple surfaces, it is desirable to excite the upper layer from above and the lower layer from below in order to reduce photobleaching. Thus, a component on a first outer layer adjacent to the support structure may be irradiated from the first side, while irradiation from the other outer side may be used to excite a component present on a layer adjacent to the other outer side.

Fig. 7 illustrates an objective lens 92 by which radiation from the emitting biological components 12, 14 on the first and second surfaces 18, 20, respectively, of the support structure 16 may be detected. The objective 92 may be one of the components of the focusing optics 32 described above. Although not dimensioned in the figures, fig. 7 illustrates exemplary dimensions between the objective 92 and the support structure 16. For example, the objective lens 92 is typically spaced from the upper substrate 42 of the support structure 16 by a distance of about 600 microns or more. The biological sample imaging system 10 may be configured to detect emitted radiation from the biological component 12 on the first surface 18 through a 300 micron upper substrate 42, which may be, for example, a glass material and have a refractive index Nd of 1.472. Additionally, the biological sample imaging system 10 may also be configured to detect emitted radiation from the biological component 14 on the second surface 20 through 100 microns of fluid affixed in the interior volume 46 of the support structure 16 by the 300 microns of the upper substrate 42.

In certain embodiments, the objective 92 may be designed for diffraction-limited focusing and imaging only on one of the first or second surfaces 18, 20 of the support structure 16. For example, in the full description of fig. 7-14, the objective lens 92 may be designed for pre-compensation of a 300 micron upper substrate 42 plus a 100 micron fluid readout buffer in the interior volume 46 of the support structure 16. Under this assumption, diffraction limited performance is only possible on the second surface 20. Furthermore, when imaging from the first surface 18, the spherical aberration introduced by the 100mm readout buffer can severely affect the quality of the imaging. However, reducing the channel thickness of the interior volume 46 of the support structure 16 may increase surface-to-surface crosstalk. Therefore, most solutions may be to correct aberrations. As such, it is desirable to use compensators that are capable of achieving diffraction limited imaging performance on the first and second surfaces 18, 20 of the support structure 16.

It should be noted that when an objective lens 92 with a high Numerical Aperture (NA) value is used, it is desirable that the compensator can have more significant effect. An exemplary high NA range in which the present invention is particularly effective includes NA numbers of at least about 0.6. For example, the NA may be at least about 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or higher. Those skilled in the art will appreciate that NA is dependent on the refractive index of the medium in which the lens is functioning, and may be higher, including, for example, 1.0 for air, 1.33 for pure water, and higher for other media such as oil. The compensator may also find application in objectives with lower NA than the examples described above. Generally, the NA value of the objective 92 is a measure of the angular size of the light that the objective can accept. For a fixed magnification, the higher the NA value, the more light the objective lens can collect. This is because the collection efficiency and resolution are improved. Therefore, when the objective lens 92 having a higher NA value is used, since it is possible to have a higher feature density, it is possible to distinguish a plurality of objects more. Thus, generally, a higher NA value for the objective 92 is beneficial for imaging. However, as the NA value increases, its sensitivity to variations in thickness of the focused and imaged through media also increases. In other words, a lower NA objective 92 has a longer depth of field and is generally not sensitive to variations in the thickness of the imaged through-medium.

Fig. 8 is an exemplary graph 94 of spherical aberration (in wavenumbers) versus thickness of the upper substrate 42 of the support structure 16 of fig. 7 in accordance with the present invention. In particular, the upper line 96 of the graph represents the magnitude of the spherical aberration of an image acquired by the biological component 12 on the first surface 18 of the support structure 16, while the lower line 98 of the graph represents the magnitude of the spherical aberration of an image acquired by the biological component 14 on the second surface 20 of the support structure 16. In the illustrated embodiment, for example, the spherical aberration generated by a 100 micron read buffer is about 4 waves, which is much higher than the diffraction limited feature that requires less than 0.25 waves. As illustrated, at 300 microns (i.e., the thickness of the upper substrate 42), the spherical aberration for the first surface 18 is approximately negative 13.2 waves (i.e., point 100), and the spherical aberration for the second surface 20 is approximately negative 17.2 waves (i.e., point 102). Fig. 9A illustrates exemplary imaging in accordance with the present invention in which it is desired that the first and second surfaces 18, 20 of the support structure 16 conform to the thickness of the upper substrate 42 (i.e., 300 microns), wherein the imaging system optimizes the second surface 20 (pre-compensates for the negative 17.2 waves). As illustrated, the imaging system can provide high imaging quality on the second surface 20, since this can be designed to do so against this background. However, the images acquired by the first surface 18 contain aberrations.

To balance the spherical aberration, it is beneficial to introduce additional thickness between the objective lens 92 and the support structure 16 (i.e., by introducing additional cover slip). For example, returning to FIG. 8, if an additional thickness of about 40 microns is introduced between the objective 92 and the first and second surfaces 18, 20 of the support structure 16, the difference between the spherical aberrations at the design thickness (i.e., 300 microns of upper substrate plus 100 microns of fluid) can be separated so that the resulting images of both the first and second surfaces 18, 20 can be of similar quality. For example, as illustrated, at 340 microns (i.e., the upper substrate 42 plus an additional 40 microns of thickness), the spherical aberration of the first surface 18 is approximately minus 15.2 waves (i.e., point 104) and the spherical aberration of the second surface 20 is approximately minus 19.2 waves (i.e., point 106), with a separation difference of approximately minus 17.2 waves (i.e., point 108), which may be characteristic of the design point of the objective 92. Fig. 9B illustrates an exemplary image in accordance with the present invention in which it is desired that the first and second surfaces 18, 20 of the support structure 16 correspond to the upper substrate 42 plus an additional thickness (i.e., 300 microns plus 40 microns), illustrating the additional thickness so as to allow a balance between the images acquired at the first and second surfaces 18, 20 of the support structure 16.

However, it is not desirable for all uses of the imaging system set forth herein to merely introduce additional thickness between the objective 92 and the support structure 16. For example, as illustrated in fig. 9A and 9B, by simply introducing an additional 40 microns of thickness between the objective 92 and the support structure 16, the resulting image of the first and second surfaces 18, 20 still experiences residual aberration generated by the design point 108 of the objective 92. Thus, a more accurate solution is to introduce additional thickness only when radiation from the biological component 12 on the first surface 18 of the support structure 16 is detected. In this context, spherical aberration consistent with the design point 108 of the objective 92 can generally be achieved by the first and second surfaces 18, 20. It should be noted that the specific dimensions and measurements discussed with reference to fig. 9A and 9B (i.e., thickness, spherical aberration values, and others) are merely exemplary ways to attempt to function as the invention. As such, these dimensions and measurements are not intended to be limiting. Indeed, the specific geometry and resulting measurement values may vary between embodiments.

For example, fig. 10A illustrates an exemplary objective 92 for imaging the second surface 20 of the support structure 16 without the aid of the compensator 110 according to the present invention. Without the compensator 110, the objective 92 can focus and direct the image of the second surface 20 of the support structure 16 according to its design and experience the design spherical aberration. However, FIG. 10B illustrates an exemplary objective 92 for imaging the first surface 20 of the support structure 16 with the aid of the compensator 110 in accordance with the present invention. By using the compensator 110 (i.e., similar to the additional 40 micron thickness discussed with reference to fig. 8 and 9), the objective 92 can focus and direct the imaging of the first surface 18 of the support structure 16 under conditions similar to the design point of the second surface 20 of the support structure 16. Thus, by detecting the imaging of the second surface 20 without the compensator 110 and detecting the imaging of the first surface 18 with the compensator 110, the objective 92 can detect the imaging of both surfaces with diffraction limited performance similar to the design of the objective 92.

The color change curve may be limited to a wavelength range between 530nm and 780 nm. Color variations of different color wavelength bandwidths may be compensated for by focusing optics 32 within the respective bandwidths. The compensator 110 should preferably be "invisible" to the focusing optics 32. In other words, the compensator 110 should correct for the spherical aberration difference of the read buffer, but should keep the color variation going towards the wavelength range of 530 to 780 nm. More specifically, the color change relationship between the peak wavelengths of 560nm, 610nm, 687nm, and 720nm should be maintained. In addition, other indicators, including NA, scene curvature, scene distortion, detection magnification, and others, should also be maintained. In addition, the compensator 110 package should be relatively small (i.e., total thickness no greater than 10 mm). Also, the positioning error of the compensator 110 is preferably insensitive.

Various designs may be used to implement the introduction of the corrective optics of compensator 110 into the optical train of the imaging optics of biological specimen imaging system 10. For example, FIG. 11 is an exemplary compensator 110 design, combining a first objective 92 and a second objective 112 for replacing the first objective 92 in an optical train in accordance with the present invention. In the illustrated embodiment, each objective 92, 112 may contain the optics necessary to image each surface, e.g., the first and second surfaces 18, 20 of the support structure 16. For example, the first objective 92 may include imaging optics necessary for focusing and imaging the emitted biological components 14 on the second surface 20 of the support structure 16, while the second objective 112 may include imaging optics plus correction optics necessary for focusing and imaging the emitted biological components 12 on the first surface 18 of the support structure 16. In operation, the first objective 92 may detect an image of the second surface 20 of the support structure 16. The first objective 92 may be replaced with a second objective 112 of the optical train, which may in this regard detect imaging of the first surface 18 of the support structure 16. The improvement of the embodiment illustrated in fig. 11 may be that it is separable and separately operable. However, a disadvantage in some cases is that having two completely separate objectives 92, 112 may not be an economical way, as some components may be duplicated for each objective 92, 112. Further, in embodiments where multiple images of an object are acquired, the use of two objective lenses may increase the computational resources required for alignment between the images. In a particular embodiment, the imaging of both surfaces may be produced by the same objective lens, providing particular advantages as set forth below. In other words, the first objective 92 need not be removed or replaced with the second objective 112 for imaging of a different surface.

FIG. 12 is another exemplary compensator 110 design that includes a corrective device 114 interposed between the objective 92 and the support structure 16 in accordance with the present invention. The correction device 114 may be, for example, a cover slip or other thin sheet of glass. As illustrated, the correction device 114 may be inserted into or removed from the optical path between the objective 92 and the support structure 16 depending on the particular surface 16 being imaged. For example, the correction device 114 may be removed from the optical path when the objective lens 92 is used to focus and image the emitting biological components on the second surface 20 of the support structure 16. Instead, the correction device 114 may be inserted into the optical path when the objective 92 is used to focus and image the emitting biological component on the first surface 18 of the support structure 16. The improvement of the illustrated embodiment shown in fig. 12 is quite evident. The required additional compensator thickness can simply be inserted in the optical path. Typically, the correction device 114 may be arranged such that it is not in physical contact with the support structure 16 or the objective lens 92.

FIG. 13 is another exemplary compensator 110 design that includes a correction ring 116 in accordance with the present invention. In the illustrated embodiment, the correction loop 116 can compensate between two states. For example, the first state 118 may correspond to the objective lens 92 focusing and detecting imaging on the second surface 20 of the support structure 16, while the second state 120 may correspond to the objective lens focusing and detecting imaging on the first surface 18 of the support structure 16. As such, the imaging optics in the objective 92 may not include the correction optics in the optical path when the correction ring 116 is in the first state 118. Conversely, when the correction ring 116 is in the second state, the imaging optics in the objective 92 may include correction optics in the optical path. Although the illustrated embodiment includes only two states 118, 120, in fact, correction ring 116 may include multiple states, for example, when imaging using more than two surfaces of support structure 16, correction ring 116 may be configured to compensate between the multiple states so that the imaging and correction optics may vary with each surface of support structure 16. The improvement of the embodiment illustrated in fig. 13 is that it is relatively easy to operate. For example, the correction ring 116 may be simply adjusted between states corresponding to different surfaces of the imaged support structure 16.

FIG. 14 is another exemplary compensator 110 design, according to the present invention, including an infinite space compensator 122. This embodiment is somewhat similar to the embodiment of the correction device 114 illustrated in FIG. 12, in that the infinite space compensator 122 can be inserted into or removed from the optical path. However, the main difference between the various embodiments is that in the embodiment shown in FIG. 14, there may be more space available (e.g., up to 10mm instead of the 600 microns illustrated in FIG. 12) for inserting the infinity space compensator 122 into the optical path. Thus, the embodiment shown in FIG. 14 allows for greater flexibility than the correction device 114 embodiment shown in FIG. 12.

In addition to the embodiments presented in fig. 11 to 14, other compensator 110 designs may prove beneficial. For example, a fluidic corrector may be interposed between the objective 92 and the support structure 16. In a fluid-type corrector design, the fluid-type corrector may be filled with a fluid to have the same effective function as the compensator 110. The optical structuring fluid matches the upper surface of the support structure 16 and matches the lower surface of the support structure 16 in the absence of fluid air. Such a design may advantageously prove to facilitate automation, as fluid may simply be inserted into or removed from the fluidic corrector depending on the surface being imaged.

Regardless of the particular embodiment chosen, all embodiments disclosed herein are characterized by the ability for repeatability and automatic use of the embodiment. An important consideration in the embodiments is the use of automated means to detect the imaging of biological components on the multiple surfaces 18, 20 of the support structure 16. This allows not only an increase in the number of images, but also provides greater flexibility in switching between multiple surfaces, depending on the requirements of the particular imaging required.

As discussed in detail above, support structures 16 that are useful in the devices or methods set forth herein may have two or more surfaces to which biological components are bound. In a particular embodiment, the surface is a fabricated surface. Any of the surfaces known in the art may be used, including but not limited to, to form the above-described array. Examples include glass, silicon, polymer structures, plastics, and the like. Particularly effective surfaces and flow cells are as discussed in PCT publication No. wo 20071123744 and are incorporated herein by reference. The surfaces of the support structure may have the same or different properties. For example, in the embodiment illustrated in FIG. 3, substrate 42 may be transparent to the excitation and emission wavelengths used in the detection method, while substrate 44 may be transparent or opaque to the excitation or emission wavelengths. Thus, the surfaces may be made of the same material, while two or more surfaces may be made of different materials.

Support structures having two or more surfaces may be made by adhering the surfaces to each other or to other support structures. For example, an adhesive material such as epoxy is applied in paste form on a planar substrate, in a pattern to form one or more channel characteristics of the flow cell. Fig. 15 shows an exemplary flow cell 124. The desired bond pattern is designed and placed on a planar surface beneath the substrate 128 using a programmable automated bond applicator, such as Millennium0M-2010, manufactured by Asymtek, inc (Carlsbad CA). The thickness of the flow cell (and the height of the intersection in the flow channel) can be set by a precision mechanical spacer 130 placed between the lower substrate 128 and the upper substrate 132. Fig. 16 shows another exemplary flow cell 134. To form a multi-layer cell, an intermediate transparent substrate layer 136 may be included that is shorter in length than the lower and upper substrate layers 128, 132. The shorter length allows fluid to contact both or all layers through the holes 138 through only one substrate. This intermediate layer 136 bifurcates the flow cell cavity horizontally and forms only two effective surface areas for adhesion of molecules of biological interest.

An exemplary method 140 for making such a flow cell is shown in fig. 17. A planar substrate is provided that serves as the structural basis for the above-described cell (block 142). The channel features required for the above-described unit are designed, for example, using a computer-aided design program (block 144). The pattern designed in this way can be exported into a file compatible with the system used to drive the automated bond application (block 146). A process is performed to apply adhesive to the substrate in a desired pattern (block 148). Before and after the adhesive is applied, precision mechanical spacers are placed on the base substrate (block 150). Subsequently, a second transparent substrate is placed on the bonding pattern and pressed down until the lower substrate is fully in contact with the mechanical spacers (block 152). A weight or other force is applied to the upper substrate to bring it into full contact with the adhesive. The spacers typically have a height that is equivalent to or slightly less than the height of the adhesive layer so that the bond can be formed without causing any adverse aberrations in the channel feature shape. The steps for adhering the substrate may be repeated depending on the number of layers desired. Alternatively, the assembly may be heat treated, for example, using an oven or UV light exposure, depending on the curing requirements of the adhesive.

Another exemplary method for making a flow cell is to use an intermediate layer to cut the desired pattern instead of a tie layer. A particularly useful material for the intermediate layer is silicon. The silicon layer may be heated to bond the lower substrate 128 and the upper substrate 132. An exemplary method of using Bisco silicon HT 6135 as an interlayer is discussed by Grover et al (see Sensors and activators B89: 315-.

Additionally, FIG. 18 illustrates an embodiment employing one radiation source and two detectors. Radiation from radiation source 22 may be directed by directing optics 30 to focusing optics 32. The focusing optics 32 irradiate the excitation radiation 58 to the biological component 12 on the first surface 18 of the support structure 16. The biological component 12 emits a fluorescent emission 60 that returns through the focusing optics 32 to the directing optics 30. This backward beam allows for the realization of multiple color channels by directing the optics 30 to the detection optics 34, which in the illustrated embodiment may include a wavelength filter 156 or some other device for separating the backward beam, and first and second color filters 158, 160. The wavelength filter 156 may separate the backward beam into two beams, one of which is directed to the first detector 36 through the first color filter 158 and the other of which is directed to the second detector 162 through the second color filter 160. In this way, the biological sample imaging system 10 can scan the first and second surfaces 18, 20 sequentially, first scanning the first surface 18 of the support structure 16 using the first excitation radiation 58 and the returned first fluorescent emissions 60 from the radiation source 22 (shown in the left half of fig. 18), and then scanning the second surface 20 of the support structure 16 using the second excitation radiation 62 and the returned second fluorescent emissions 64 from the same radiation source 22 (shown in the right half of fig. 18).

Additionally, FIG. 19 illustrates an embodiment using two radiation sources and two detectors. Both surfaces 18, 20 of the support structure 16 may be scanned in a sequenced manner. However, in this embodiment, the first surface of support structure 16 is first scanned using first radiation source 22 that produces first excitation emissions 58 and first fluorescent emissions 60 (shown in the left half of FIG. 19), while the second surface 20 of support structure 16 is scanned using second radiation source 24 that produces second excitation emissions 62 and second fluorescent emissions 64 (shown in the right half of FIG. 19).

In the above-described embodiment, the scanning of the first and second surfaces 18, 20 of the support structure 16 may be performed sequentially, and the steps of scanning the first and second surfaces 18, 20 of the support structure 16 may be performed in a variety of ways, for example, it is possible to scan a single line of the first surface 18, then a single line of the second surface 20, and then gradually move the first and second surfaces 18, 20 relative to the excitation radiation 58, 62 by translating the support structure 16, the directing optics 30, the focusing optics 32, or a combination thereof, so as to repeat the steps of scanning the lines. Alternatively, the entire area of the first surface 18 may be scanned before the area of the second surface 20 is scanned. The various processing steps may be taken depending on several variables including the particular configuration of the biological component sites 12, 14 on the surfaces 18, 20, as well as other variables including environmental and operating conditions.

Particular embodiments may allow multiple surfaces of the support structure 16 to be excited simultaneously. For example, fig. 20 illustrates an embodiment using two radiation sources and two detectors. However, in this embodiment, the first surface 18 and the second surface 20 of the support structure 16 may be scanned simultaneously. This can be done using focusing lenses 164, 166, 168, 170 and dichroic mirrors 172 along the excitation path to switch the multiple surfaces and filters 158, 160 to achieve multiple color channels. The illustrated embodiment can then also be extended to any number of detectors to improve throughput, scanning efficiency, and reduce movement of filters and other system components.

Fig. 21 illustrates another embodiment using two radiation sources and two detectors, which allows for simultaneous scanning of the first and second surfaces 18, 20 of the support structure 16. However, in the illustrated embodiment, not only are focusing lenses 164, 166, 168, 170 and dichroic mirror 172 used in the excitation path, but focusing lenses 174, 176 may be used upstream of the first and second detectors 36, 162, in combination with filters 158, 160 along the emission path to switch surfaces and implement multiple color channels. The illustrated embodiment can then be extended to use any number of detectors to improve throughput and scanning efficiency.

For example, fig. 22 illustrates an embodiment using multiple radiation sources and multiple detectors to enable simultaneous output of multiple channels with few moving parts. In the illustrated embodiment, radiation sources 22 and 24 can be replaced by radiation source groups 178 and 180, which can output multiple radiation sources and varying wavelengths. Additionally, in the illustrated embodiment, the detectors 36 and 38 may be replaced by detector sets 182 and 184. These detector sets 182, 184 are also capable of detecting multiple color channels. This embodiment thus illustrates that the considerable applicability of the technique of the present invention can be extended to the range of configurations of imaging assemblies on multiple surfaces of the support structure described above.

In the above-described embodiments, the scanning of the first and second surfaces 18, 20 of the support structure 16 may be performed simultaneously, and the focusing of the source of excitation radiation 58 may be accomplished in a variety of ways. For example, it is possible to focus the excitation radiation 58 preferentially on one surface over the other. In fact, this is required due to the nature of the configuration of the first surface 18 relative to the second surface 20. However, other focusing techniques may be employed depending on the particular configuration of the support structure 16. Furthermore, these various configurations facilitate imaging the upper surface (i.e., the surface closer to the radiation source) first to reduce photo-bleaching of the surface by the compensator, which results from imaging the lower surface (i.e., the surface further from the radiation source) first. This process of imaging surface selection can be applied both when the surfaces are imaged sequentially and when the surfaces are imaged simultaneously.

In addition, the above embodiments have described an epi-fluorescence imaging scheme in which the excitation emission is directed to the surface of the support structure 16 from above and receives return fluorescence radiation from the same side. However, the present techniques may be extended to other configurations as well. For example, these techniques may also be used in conjunction with TIR imaging to direct radiation from the side to illuminate the surface of the support structure at a range of oblique angles to deliver excitation radiation within the support structure or to deliver radiation into the support structure from a prism around it. The use of TIR techniques is discussed in U.S. patent publication No.2005/0057798, which is incorporated herein by reference. This technique facilitates fluorescent emission from the component to pass out for imaging while reflected excitation radiation is output through the other side, as opposed to through that side as it enters, where biological components on multiple surfaces can be imaged again sequentially or simultaneously.

For example, in fig. 23, a TIR biological sample imaging system 186 is shown. A support structure 188 may be used that includes a plurality of flow channels 190 containing biological components. For example, the support structure 188 may be a flow cell through which reagents, cleaning solutions, and other fluids may be introduced using the flow channel 190 to contact emissive components attached to the surface of the flow cell. The support structure 188 may be supported by a prism 192. In the TIR biological sample imaging system 186, a radiation source 194 may output a radiation beam 196 from a side of the support structure 188 through a prism 192. The radiation beam 196 may, for example, be directed to a bottom surface of one of the flow channels 190 of the support structure 188 to excite an emissive component adjacent the surface.

As will be discussed in further detail below, so long as the angle of inclination of the radiation beam 196 is within a certain angular range (as discussed in U.S. patent publication 2005/0057798), a portion of the radiation beam 196 will be reflected by the bottom surface, while a separate fluorescence emission beam from the surface-bonded emitting component will be directed to focusing optics 198. In general, a well-corrected radiation beam is used to prevent angular spread within the beam, thereby preventing unnecessary obstruction by total internal reflection. The fluorescence emission beam may be passed through focusing optics 198, directing optics 200, and detection optics 202 to direct the beam to a detector 204. The focusing optics 198, directing optics 200, detection optics 202, and detector 204 may operate in much the same manner as the epi-fluorescence technique described above. In the TIR biological sample imaging system 186, the focusing light source 206 may be used as a separate light source from the radiation source 194 to optically focus it on a particular surface to be imaged. For example, the focusing light source 206 may be directed to the directing optics 200, which in turn directs it to the focusing optics 198, which are used to focus the system on a particular surface of the support structure 188.

The TIR biological sample imaging system 186 may also include a translation system 208 for moving the support structure 188 and the prism 192 in one or more dimensions. The translation system 208 may be used to focus, redirect the radiation source 194 on different areas of the support structure 188, and to move the support structure 188 and the prism 192 to the heating/cooling station 210. The heating/cooling station 210 may be used to heat and cool the support structure 188 before and after imaging. In addition, a control/processing system 212 may be used to control the operation of the radiation source 194, the focusing light source 206, and the heating/cooling station 210, to control the movement and focusing of the focusing optics 198, the translation system 208, and the detection optics 202, and to control the signal acquisition and processing of the detector 204.

As described above, the TIR method of imaging may be used to direct a radiation beam 196 from the side of the support structure 188, as illustrated in fig. 24. Each flow channel 190 of the support structure 188 may include a bottom surface 214 and an upper surface 216, and the emissive components may be selectively adhered to one or both surfaces. In the illustrated embodiment, the radiation beam 196 is directed to a bottom surface 214 of one of the flow channels of the support structure 188. A portion of the radiation beam 196 may be reflected from the bottom surface 214 of the flow channel 190, as indicated by the reflected light source 218. However, as long as the angle of inclination of radiation beam 196 is within a certain angular range, a separate fluorescence emission beam 220 may be emitted from the emission component to focusing optics 198, which in the illustrated embodiment is lens objective 222. Indeed, directing radiation beam 196 to bottom surface 214 of flow channel 190 of support structure 188 is a typical implementation of TIR imaging methods. In so doing, however, image data collected from the upper surface 216 of the flow channel 190 of the support structure 188 may be ignored.

Accordingly, the orientation of the radiation source 194 and/or the support structure 188 and the prism 192 may be adjusted to allow the radiation beam 196 to no longer be directed onto the bottom surface 214 of the flow channel 190 of the support structure 188, as illustrated in fig. 25. In the illustrated embodiment, the radiation beam 196 is oriented such that the radiation beam 196 passes through the prism 192 and the support structure 188 until the air/glass interface 224 of the support structure 188 is terminated, at which point the radiation beam 196 is redirected onto the upper surface 216 of the flow channel 190 of the support structure 188. In this regard, a portion of the radiation beam 196 may be reflected to another air/glass interface 224 of the support structure 188. However, a separate fluorescent emission beam 220 may be emitted from an emitting component on the upper surface 216 to the lens objective 222. Using this technique, the upper surface 216 of the flow channel 190 of the support structure 188 may be imaged using TIR imaging methods. This may in effect allow for twice the image data output for cluster-based sequencing applications, while also keeping other variables the same, such as surface coverage, cluster creation, and sequencing.

To accomplish TIR imaging of the upper surface 216 of the flow channel 190 of the support structure 188, the radiation beam 196 reaches the air/glass interface 224 of the support structure 188 in a non-perturbing manner. In so doing, the radiation beam 196 does not first contact the emitting component adjacent the flow channel 190. In doing so, either radiation source 196 may be directed adjacent to flow channel 190 or adjacent to flow channel 190 may be index matched to the support structure 188 material. In an embodiment, the flow channels 190 may be spaced apart from each other in the support structure 188, leaving sufficient space between the flow channels 190 for the radiation beam 196 to pass through. However, the flow channel 190 partitioned in this way can significantly reduce the number of transmit components used for imaging. Thus, in other embodiments, it is possible to achieve the same effect by filling other flow channels 190 with index matched fluids. Doing so may allow the radiation beam 196 to be more easily directed to the upper surface 216 of the flow channel 190 of the support structure 188.

In this way it is also possible to direct the radiation beam 196, which reflects off of the multiple upper surfaces 216 of the flow channel 190 of the support structure 188, as illustrated in fig. 26. To accomplish this, the spacing of the flow channels 190 may be matched to the angle of the radiation beam 196 such that the radiation beam 196 is able to pass through the flow channels 190 to reach the air/glass interface 224 of the support structure 188 in a non-perturbing manner while also being able to reflect back and forth between the upper surface 216 of the flow channels 190 and the air/glass interface 224 of the support structure 188. In some embodiments, mirrors 226 or other reflective suitable materials may be used within some of the flow channels 190 to facilitate this multiple reflection technique. In any event, given the N flow channels 190, it is only possible to image the upper surfaces 216 of the N-2 flow channels 190 in this manner due to the fact that the outer flow channels 190 on the other side of the support structure 188 cannot be involved using these techniques. However, modifications of the prism 192 and/or the support structure 188 may allow imaging of the upper surface 216 of the outermost flow channels 190. For example, the support structure 188 may be designed to be placed in the prism 192 to allow the radiation beam 196 to pass through the sides of the support structure 188.

In some embodiments, as briefly described above with reference to fig. 23, the support structure 188 may be moved to a heating/cooling station 210, for example, by operation of the translation system 208. The heating/cooling station 210 may constitute a heating and cooling support structure 188 before and after imaging. In fact, the heating/cooling station 210 may constitute an upper surface 228 and a bottom surface 230 of the heating and cooling support structure 188, as illustrated in fig. 27. Indeed, all surfaces of the support structure 188 may be heated or cooled at a heating/cooling station. In this manner, it is also possible to heat and cool the upper surface 216 and the bottom surface 214 of the flow channel 190 of the support structure 188 by directly contacting one or more surfaces of the flow channel with a heating or cooling device. Of course, this may facilitate the development of biological components in the flow channels of the support structure 18 and thus imaging. Although the use of heating/cooling station 210 has been presented herein with respect to TIR imaging methods, heating/cooling station 210 may also be used to heat and cool multiple sides of a support structure used in conjunction with the epi-fluorescence imaging methods discussed herein.

In particular embodiments, the present invention uses sequencing-plus-synthesis (SBS) methods. In SBS, four fluorescently labeled modified nucleotidyl transferases are used to determine the sequence of the nucleotidyl transferase that presents nucleic acids on the surface of a support structure such as a flow cell. Exemplary SBS systems and methods that can be employed with the devices and methods set forth herein are as described in U.S. patent nos. 7057026; U.S. patent publication Nos.2005/0100900, 2006/0188901, 2006/0240439, 2006/0281109 and 2007/0166705; and PCT publication Nos. WO05/065814, WO06/064199 and WO 07/010251; this is incorporated by the application.

In particular uses of the devices and methods set forth herein, flow cells containing arrayed nucleic acids are processed through large, several cycles of repetition throughout the sequencing process. The nucleic acid is prepared such that it comprises a oligo (poly) nucleotide substrate close to the unknown target sequence. To initiate the SBS sequencing cycle, one or more differently labeled nucleosides and a DNA polymerase are flowed into the flow cell. A single nucleoside can be added at once, or the nucleosides used in the sequencing process can be specifically designed to possess reversible termination properties, thus allowing each cycle of the sequencing reaction to occur simultaneously in the presence of all four labeled nucleosides (A, C, T, G). Following nucleoside addition, features on the surface can be imaged to determine the identity of the bound nucleoside (based on labeling of the nucleoside). Subsequently, reagents are added to the flow cell to remove the blocked 3' terminus (if necessary) and to remove the labels from the respective binding substrates. The cycle is then repeated and the sequence of each cluster is read over multiple cycles of the chemical reaction.

Other sequencing methods may also be used, which use a cycle reaction method and each cycle involves dropping one or more reagents into the nucleic acid on the surface and imaging the surface of the bound nucleic acid, for example, using bundled sub-sequencing and sequencing. Useful sub-sequencing reactions are discussed in U.S. Pat. No.7,244,559 and U.S. patent application publication No.2005/0191698, incorporated herein by reference. Sequencing of the binding reaction is discussed by Shendure et al (see Science 309: 1728-1732(2005)) and U.S. Pat. Nos.5,599,675 and 5,750,341, which are incorporated herein by reference.

The methods and devices discussed herein are also useful for detecting ostensibly occurring features used in genotyping assays, expression analysis and other assays well known in the art, as discussed in U.S. patent application publication Nos.2003/0108900, US 2003/0215821 and US 2005/0181394, which are incorporated herein by reference.

While certain features of the invention have been illustrated and discussed herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (19)

1. A method for imaging a biological sample, comprising:

(a) detecting radiation emitted by a first emissive component of a biological sample disposed on a first surface of a flow cell using a detector, wherein the flow cell is mounted on an imaging station;

(b) inserting correction optics between the detector and the flow cell;

(c) detecting radiation emitted by a second emissive component of the biological sample disposed on a second surface of the flow cell using a detector and correction optics, wherein the first and second surfaces are in an arrangement such that one of the two surfaces is disposed between the detector and the other of the two surfaces, wherein the correction optics reduce detection aberrations at the one of the two surfaces due to the arrangement; and

(d) repeating steps (a) through (c) while maintaining the flow cell on the imaging station.

2. The method of claim 1, wherein the first surface is disposed between a detector and a second surface.

3. The method of claim 2, wherein a fluid is between the two surfaces and the corrective optics reduce aberrations due to the fluid.

4. The method of claim 3, wherein the detector is initially configured for diffraction limited detection on the second surface and the corrective optics are configured for diffraction limited detection on the first surface.

5. The method of claim 3, wherein the aberrations comprise spherical aberrations.

6. The method of claim 1, wherein the first surface is disposed between a radiation source and a second surface.

7. The method of claim 1, wherein step (b) comprises inserting a calibration device between the objective lens and the flow cell, wherein the objective lens is disposed between the detector and the flow cell.

8. The method of claim 1, wherein step (b) comprises inserting a corrective lens between the detector and the objective lens, wherein the objective lens is disposed between the detector and the flow cell.

9. The method of claim 1, wherein steps (a) and (c) comprise acquiring images of the surfaces.

10. The method of claim 1, wherein steps (a) and (c) comprise focusing the radiation onto an area on the first or second surface containing the first or second emissive component.

11. The method of claim 1, wherein the emitted radiation is detected using epi-fluorescence excitation.

12. The method of claim 1, wherein the excitation radiation is directed to the first and second surfaces by total internal reflection excitation.

13. The method of claim 12, wherein the excitation radiation and flow cell are configured to generate total internal reflection for exciting the first surface and the second surface.

14. The method of claim 1, wherein the first and second emissive components comprise fluorescently labeled nucleic acids of a nucleic acid sample.

15. The method of claim 1, wherein the first and second emissive components comprise separate features in an array of features on the first and second surfaces, respectively.

16. The method of claim 15, wherein the separate features each comprise a population of identical molecules.

17. The method of claim 1, wherein the color shift curve of the radiation detected in step (a) is the same as the color shift curve of the radiation detected in step (b).

18. The method of claim 1, wherein the field curvature detected in step (a) is the same as the field curvature detected in step (b).

19. The method of claim 1, wherein the detected field distortion in step (a) is the same as the detected field distortion in step (b).