HK1218671B - A microscope module for imaging a sample - Google Patents

Description

Microscope module for sample imaging

CROSS-REFERENCE TO RELATED APPLICATIONS

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Technical Field

The present invention relates to the field of microscope modules for imaging samples.

Background Art

Selective plane illumination microscopy (SPIM) is a technique that uses the formation of a light sheet to illuminate a sample, and is a vertical detection system that can image optical sections of a sample, which can be living or non-living. In most embodiments, the SPIM system requires extensive sample preparation to keep the sample in the correct position for imaging. For example, the sample is generally embedded in an agarose cylinder, which is immersed in a chamber filled with an immersion medium (such as water). People have known about this technology for over a hundred years, but it has only recently found widespread application in imaging biological samples. One disadvantage of this technology is that agarose is not compatible with all biological specimens. In current SPIM systems, the sample is also embedded in a vertical cylinder of agarose of a limited height. This arrangement does not allow access to the sample during imaging or repositioning of the sample. Such an arrangement limits the number of samples that can be imaged because, for example, it is impossible to stack 50 samples within the limited length of the agarose cylinder.

SPIM systems are described, for example, in International Patent Application No. WO 2004/053558 (Stelzer et al., assigned to the European Molecular Biology Laboratory). This disclosure teaches a microscope in which a thin strip of light (a light sheet) illuminates a sample (specimen) and is observed by a detector. The axis of the detector is positioned substantially perpendicular to the direction of the illumination beam. The sample is moved through the strip of light, and the detector records diffuse light from the sample or fluorescence from the sample in a series of images. A three-dimensional image of the sample can be created by optically sectioning the sample and then reconstructing the entire image of the sample.

Shroff et al. have developed a module for conventional microscopes that is coupled to the translational base of a conventional microscope (International Patent Application No. WO 2012/122027, Shroff et al., assigned to the U.S.) The combination of the module and an inverted microscope enables the same sample to be imaged in two complementary ways.

Summary of the Invention

A microscope module for imaging one or more samples is disclosed. The microscope module includes an illumination device for generating an illumination beam along an illumination beam path and at least one detection device having a detection path. The illumination beam is arranged to illuminate the lower surface of the one or more samples. The illumination beam path is arranged to form an angle with the detection path. In one aspect of the present disclosure, the angle is substantially a right angle. The sample is placed in a culture medium. It is not necessary to embed the sample in a solid or viscous mounting medium, which may be incompatible with the survival of the biological sample and also complicates the retrieval and manipulation of the sample.

The sample is placed in a sample holder. The bottom of the sample holder is at least partially transparent to the illumination beam, so that the illumination beam can illuminate the sample. An example of such a transparent bottom is a membrane. The sample holder includes at least one protrusion in which the sample is supported. In one aspect of the present disclosure, the protrusion can be in the form of an elongated groove in which a plurality of samples are supported in the culture medium.

The sample holder is arranged to be easily removable from the microscope module. This allows the sample to be cultured in the sample holder outside the microscope module and to be placed back into the microscope module for imaging.

The disclosed arrangement allows the illumination and detection objectives to be placed in an immersion medium that is separate from the culture medium containing the sample. Separating the culture medium from the immersion medium helps maintain sterility and also allows for the use of smaller volumes of culture medium. The transparent bottom, immersion medium, and culture medium have substantially the same refractive index to reduce optical aberrations.

The present disclosure also teaches a method for imaging multiple samples, comprising arranging an illumination objective to illuminate lower surfaces of the multiple samples and arranging a detection objective to detect emitted light from the multiple samples at an angle substantially perpendicular to the illumination beam path. The detected light can be used to create one or more images of the multiple samples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an overview of a prior art SPIM arrangement for sample imaging.

FIG2 shows an overview of a SPIM arrangement used in one aspect of the present disclosure.

Figure 3 shows an overview of the microscope module.

Figure 4 shows an elongated trough in which a sample is placed.

Detailed description of the invention

The present invention will now be described with reference to the accompanying drawings. It should be understood that the embodiments and aspects of the invention described herein are merely examples and do not in any way limit the scope of protection of the claims. The present invention is defined by the claims and their equivalents. It should be understood that features of one aspect or embodiment of the present invention may be combined with features of different aspects and/or embodiments of the present invention.

FIG1 illustrates the basic principle of SPIM and is described more extensively in U.S. Pat. No. 7,554,725, the disclosure of which is incorporated herein by reference. The apparatus 10 includes a laser 20 that generates a light sheet 30 that is passed through an illumination objective 25 to illuminate a section of a specimen 40. The light sheet 30 is along an illumination beam path 35. The detection objective 65 is arranged so that a detection direction 55 is approximately at right angles to the plane of the light sheet 30 (i.e., perpendicular to the illumination beam path 35).

The sample 40 can be rotated about an axis of rotation 45, and the light sheet 30 can be arranged to illuminate an optical section of the specimen 40. The laser light 20 typically excites fluorophores in the sample 40 to fluoresce in many directions.

The detector 50 detects a portion of the fluorescent light emitted from fluorophores in the sample 40 that have been excited by the radiation in the light sheet 30 via a detection objective 65 and an optical arrangement 66. The detector 50 has an imaging device 60, such as a CCD camera, connected to a processor 70 having a memory store 80. The memory store 80 stores individual images 85 for each optical section from the sample 40, and the processor 70 can create a three-dimensional image of the sample 40.

FIG2 shows an embodiment of a microscope apparatus 200 used in the present disclosure. The same reference numerals are used to indicate the same elements in FIG1 and FIG2. It is not necessary to embed the sample 40 in agarose in the present disclosure because the sample 40 is supported sufficiently stably in the apparatus, as will be explained below.

Laser 20 generates a light sheet 30 that passes through a mirror 67 and an illumination objective 25 to illuminate a section of sample 40. Light sheet 30 enters sample 40 through the lower surface of sample 40. A significant portion of the fluorescence emitted from sample 40 passes through detection objective 65, is reflected by mirror 27, and is focused by optical device 66 onto imaging device 60 in detector 50 to form an image. The image from detector 50 is transmitted to processor 70 and then stored in memory 80 as individual images 85.

FIG3 shows an example of a microscope module 300 having an illumination objective 210 and a detection objective 220. The illumination objective 210 is illuminated by an illumination beam (light sheet) along an illumination beam path 215. The illumination beam path 215 through the illumination objective 210 and the detection beam path 225 through the detection objective 220 are arranged approximately at right angles to each other. Both the illumination objective 210 and the detection objective 220 are located in an immersion medium 230, which typically comprises degassed water or immersion oil. The degassing of the water ensures that no air bubbles are present in the immersion medium 230.

The illumination beam path 215 through the illumination objective 210 is located below the sample support 240 at an angle of approximately 30° to the plane of the sample support 240. The detection path 225 is therefore positioned at approximately 60° to the plane of the sample support 240. Flexible plastic rings around the illumination objective 210 and the detection objective 220 prevent leakage of the immersion medium 230.

The sample holder 240, having walls 250, is made of a biocompatible material (such as, but not limited to, PEEK) and has a base 260 made of a thin transparent film, such as FEP film manufactured by Dupont, having a refractive index substantially similar to that in the immersion medium 230 and/or culture medium 280 to reduce optical aberrations. Thus, the transparent film in the base 260 allows radiation to pass through to the sample 270 located on the top side of the transparent film 260. The transparent film forming the base 260 is attached to the walls 250 of the sample holder 240 using a biocompatible silicone glue or by clamping. The transparent film is curved in areas not supported by the walls 250 to maintain the film under tension. The sample holder 240 is open at the top, and the opening facilitates access and removal of the sample 270, if necessary. The transparent film is plasma-treated to render it hydrophilic, thereby helping to prevent the formation of bubbles in the immersion medium 230.

In a suitable culture medium 280, sample 270 is located in the curved area in the transparent film. Culture medium 280 is an embryo or tissue culture medium and may have a layer of oil on its surface to prevent evaporation. The different refractive indices of the oil will not affect the imaging of sample 270 because the illumination beam path 215 and/or the detection path 225 do not pass through the oil. Culture medium 280 can have a very small volume, for example 10 μl. Examples of such culture medium 280 include, but are not limited to, KSOM, M16 (mouse embryo), DMEM and RPE (cell culture). It is not necessary for sample 270 to be embedded in an agarose cylinder (as known in the art). Projection 290 can be extended to form a groove (refer to Figure 4).

The microscope module 300 shown in Figure 3 is able to isolate the immersion medium 230 from the culture medium 280. This can be seen as a difference from the device 10 of Figure 1, in which the immersion medium and the aqueous medium holding the sample 40 are the same.

The sample 270 can also be easily manipulated since the sample 270 is accessible from the front through the culture medium 280. An opening in the sample holder 240 allows the sample 270 to be accessed.

3 , only the lower surface of sample 270, including the bottom and side surfaces, will be illuminated by radiation from illumination objective 210. Similarly, fluorescence from the lower surface of sample 270 will be collected by detection objective 220 and thus used to create image 85 in memory storage 80.

The protrusion 290 may be in the form of an elongated slot 295, as shown in Figure 4. This aspect of the invention allows multiple samples of the sample 270 to be placed along the slot and imaged using the same microscope module 300. This arrangement would allow high throughput imaging of multiple samples 270.

The microscope module 300 enables long-term high-throughput live cell and embryo imaging experiments, such as in vitro imaging of mammalian embryos and oocytes.

A method for conducting long-term, high-throughput live cell and embryo imaging experiments can be performed using a microscope module 300. The method includes configuring an illumination objective 210 so as to generate an illumination beam to illuminate the lower surface of a plurality of samples 270 along an illumination beam path 215. The detection objective 220 collects a portion of the fluorescence emitted from the plurality of samples 270. Fluorescence is emitted in all directions, and fluorescence in an arc of approximately 120° around a detection path 225 will be collected. The fluorescence collected by the detection objective 220 is reflected by a reflector 27 and focused by an optical device 66 onto an imaging device 60 in a detector 50. The imaging device 60 sends data associated with an image 85 to a processor 70, and the processor 70 is capable of creating a three-dimensional image of one or more of the plurality of samples 270.

4 , the elongated slot 295 can be moved so that the detection objective lens 220 and the illumination objective lens 210 scan the elongated slot 295 to image different samples of the plurality of samples 270. The detection objective lens 220 and the illumination objective lens 210 remain fixed to the optical platform.

The culture medium 280 remains intact through either the detection objective or the illumination objective and remains sterile allowing for long term experiments.

Reference numerals

10 Devices

20 Laser

25 Illumination objective

27 Reflection Realm

30 light sheets

35 Illumination beam path

40 samples

45 Rotation axis

50 detectors

55 Detection direction

60 Imaging Equipment

65 Detection objective lens

66 Optical Devices

67 Reflection Realm

70 processors

80 Memory Storage

85 images

200 Microscope Device

210 Illumination Objective

215 Illumination beam path

220 Detection objective lens

225 Detection Path

230 impregnation medium

240 Sample Holder

250 wall

260 bottom

270 samples

280 culture medium

290 protrusion

295 slots

300 Microscope Module

Claims (13)

1. A microscope module (300) for imaging a sample (40, 270) in a microscope apparatus (200), the microscope apparatus (200) comprising: - A light source (20) for generating the illumination beam, and -A detector (50) for detecting the emitted light, The microscope module (300) includes: - Sample holder (240), which is removably disposed in the microscope apparatus (200) and adapted to support a sample (40, 270) in the sample holder (240), and the sample holder (240) has a bottom (260) that is at least partially transparent to the illumination beam; - At least one illumination objective (210) located below the sample holder (240) and arranged in the microscope apparatus (200) to guide the illumination beam along the illumination beam path (215) through the bottom (260) of the sample holder (240) to illuminate the sample (40, 270); - At least one detection objective (220), said at least one detection objective (220) being located below the sample holder (240) and arranged in the microscope apparatus (200) to collect light emitted from the sample (270) along a detection path (225) at an angle to the illumination beam path (215) and passing through the bottom (260) of the sample holder (240). The illumination objective (210) and the detection objective (220) are located in an immersion medium (230) that is in contact with at least a partially transparent bottom (260) of the sample holder (240). 2. The microscope module (300) according to claim 1, wherein the angle between the detection path (225) and the illumination beam path (215) is substantially right-angled. 3. The microscope module (300) according to claim 1, wherein the refractive index of the immersion medium (230) is substantially similar to the refractive index of the at least partially transparent bottom (260) of the sample holder (240). 4. The microscope module (300) according to claim 1, wherein at least a partially transparent bottom (260) of the sample holder (240) is made of a membrane. 5. The microscope module (300) according to claim 1, wherein the sample (40, 270) is in a culture medium (280). 6. The microscope module (300) according to claim 5, wherein the refractive index of the culture medium (280) is substantially similar to the refractive index of the at least partially transparent bottom (260) of the sample holder (240). 7. The microscope module (300) according to claim 1, wherein at least a partially transparent bottom (260) of the sample holder (240) includes a protrusion (290) in which the sample (270) is supported. 8. The microscope module (300) according to claim 7, wherein the illumination beam is arranged to illuminate the sample (270) through the bottom (260) of the protrusion (290). 9. The microscope module (300) according to claim 8, wherein the protrusion (290) is elongated. 10. The microscope module (300) according to claim 1, wherein the illumination beam path is arranged substantially at 30° to the horizontal position. 11. An imaging method for one or more samples (40, 270), comprising: One or more samples (40, 270) are placed in a sample holder (240) having a bottom (260) that is at least partially transparent; A sample holder (240) is removably disposed above an illumination objective (210) and a detection objective (220), wherein the illumination objective (210) and the detection objective (220) are located in an immersion medium (230) that is in contact with at least a partially transparent bottom (260) of the sample holder (240); An illumination beam along the illumination beam path (215) is generated by passing light from the light source (20) through the illumination objective (210); One or more samples (40, 270) are illuminated by an illumination beam through at least a partially transparent bottom (260) of the sample holder (240); The light emitted from one or more samples (40, 270) along a detection path (225) at an angle to the illumination beam path (215) is detected using a detection objective (220); and Create images of the one or more samples (40, 270). 12. The method of claim 11, wherein the sample holder (240) further comprises an elongated groove (295) along which a plurality of samples (40, 270) are placed for imaging, the method further comprising the step of selecting different samples among the plurality of samples (40, 270) in the elongated groove (295) for imaging. 13. Use of the microscope module (300) according to any one of claims 1 to 10 for imaging biological samples.