HK40013087A - A microscope module for imaging a sample - Google Patents

Description

Microscope module for imaging a sample

The application is a divisional application, the international application number of the original application is PCT/EP2014/059307, the international application date is 2014, 5, 7 and the national application number is 201480025949.5, the date of entering the national stage of China is 2015, 11, 05 and the name of the invention is 'microscope module for sample imaging'.

Technical Field

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

Background

Selective Planar Illumination Microscopy (SPIM) is a technique that employs the formation of a light sheet to illuminate a sample, which may be living or non-living, and is a vertical detection system capable of imaging an optical slice of the sample. In most embodiments, SPIM systems require extensive sample preparation to keep the sample in the correct position for imaging. For example, the sample is typically embedded in an agarose cylinder, which is immersed in a chamber filled with an immersion medium (e.g., water). This technique has been known for over a hundred years, but has not until recently found widespread use in imaging biological samples. One disadvantage of this technique is that agarose is not compatible with all biological specimens. In current SPIM systems, the samples are also embedded in agarose vertical cylinders of defined 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 not possible to stack 50 samples within the limited length of an agarose cylinder.

The SPIM system is described, for example, in International patent application No. WO 2004/053558(Stelzer et al, assigned to European molecular biology laboratories). The present disclosure teaches a microscope in which a thin strip of light (light sheet) illuminates the sample (specimen) and the sample is viewed through a detector. The axis of the detector is located in a direction substantially perpendicular to 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. Three-dimensional imaging of a sample may be created by optically sectioning the sample and then reconstructing an entire image of the sample.

Shroff et al have developed a module for a conventional microscope coupled to the translation stage of a conventional microscope (International patent application No. WO 2012/122027, Shroff et al, assigned to the United states). The combination of the module and the inverted microscope enables the same sample to be imaged in two ways complementary to each other.

Disclosure of Invention

A microscope module for imaging one or more samples is disclosed. The microscope module comprises an illumination device for generating an illumination beam along an illumination beam path and at least one detection device with a detection path. The illumination beam is arranged to illuminate the lower surface of the one or more samples. The illumination beam path is disposed at an angle to the detection path. In one aspect of the 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 carrier medium (mounting medium), which may be incompatible with the survival of the biological sample and also complicate the retrieval and manipulation of the sample.

The sample was placed in a sample holder (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. One example of such a transparent bottom is a film. The sample holder includes at least one protrusion in which the sample is held. In one aspect of the disclosure, the projections may be in the form of elongated slots in which multiple samples are supported in a medium (helld).

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

The arrangement of the present disclosure allows the illumination objective and the detection objective to be placed in an immersion medium that is separate from the medium in which the sample is placed. The separation of the culture medium from the infusion medium helps to maintain sterility and also allows the use of small volumes of culture medium. The transparent bottom, the immersion medium and the culture medium have substantially the same refractive index to reduce optical aberrations.

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

Drawings

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

Fig. 2 shows an overview of a SPIM arrangement used in one aspect of the present disclosure.

Fig. 3 shows an overview of the microscope module.

Fig. 4 shows an elongated slot in which a sample is placed.

Detailed description of the invention

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

Fig. 1 illustrates the basic principles of SPIM and is more broadly described in U.S. patent No. US 7554725, the disclosure of which is incorporated herein by reference. The apparatus 10 includes a laser 20 that produces a light sheet 30 that is passed through an illumination objective 25 to illuminate a slice of a specimen 40. Light sheet 30 follows illumination beam path 35. The detection objective 65 is arranged such that the detection direction 55 is substantially at right angles to the plane of the light sheet 30 (i.e. perpendicular to the illumination beam path 35).

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

The detector 50 detects, via the detection objective 65 and the optical device 66, the portion of the fluorescence emitted from the fluorophores in the sample 40 that have been excited by the radiation in the light sheet 30. 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 storage 80 stores a respective image 85 from each optical slice of the specimen 40, and the processor 70 may create a three-dimensional image of the specimen 40.

Fig. 2 illustrates an embodiment of a microscope device 200 used in the present disclosure. Like reference numerals are used to indicate like elements in fig. 1 and 2. It is not necessary in this disclosure to embed the sample 40 in agarose because the sample 40 is sufficiently stable to be supported in the device, as will be explained below.

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

Fig. 3 shows an example of a microscope module 300 with 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 path 225 through the detection objective 220 are arranged substantially at right angles to each other. Both the illumination objective 210 and the detection objective 220 are located in an immersion medium 230, which comprises typically degassed water or immersion oil (immersion oil). The degassing of the water ensures that air bubbles are not present in the immersion medium 230.

The illumination beam path 215 through the illumination objective 210 is located below the sample holder 240 at about 30 deg.c from the plane of the sample holder 240. The detection path 225 is therefore positioned at approximately 60 deg. to the plane of the sample holder 240. The flexible plastic ring around the illumination objective 210 and the detection objective 220 prevents leakage of the immersion medium 230.

The sample holder 240 with walls 250 is made of a biocompatible material, such as, but not limited to PEEK, and has a bottom 260 made of a thin transparent film, such as that produced by DupontAn FEP film having a refractive index substantially similar to the refractive index in the immersion medium 230 and/or the culture medium 280 to reduce optical aberrations. Thus, the transparent film in the bottom 260 allows radiation to pass onto the sample 270 on the front side (top side) of the transparent film 260. The transparent membrane forming the bottom 260 is attached to the wall 250 of the sample holder 240 by a biocompatible silicone glue (silicone glue) or by clamping. The transparent film is curved in the areas not supported by the walls 250 to hold the transparent film under tension. The sample holder 240 is open at the top and the opening allows for easy access and removal of the sample 270, if desired. The transparent film is plasma treated to make it hydrophilic, thereby helping to prevent immersionAir bubbles are formed in the immersion medium 230.

In a suitable medium 280, the sample 270 is located in a curved region in the transparent membrane. 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 do not affect the imaging of the sample 270 because the illumination beam path 215 and/or the detection path 225 do not pass through the oil. The culture medium 280 may have a very small volume, for example 10. mu.l. Examples of such media 280 include, but are not limited to, KSOM, M16 (mouse embryo), DMEM, and RPE (cell culture). It is not necessary to embed the sample 270 in an agarose cylinder (as is known in the art). The projection 290 may be elongated to form a slot (see fig. 4).

The microscope module 300 shown in fig. 3 is capable of isolating the immersion medium 230 from the culture medium 280. It can be seen that this is different from the device 10 of fig. 1, in which the immersion medium is the same as the aqueous medium holding the sample 40 in the device 10 of fig. 1.

Sample 270 can also be easily manipulated because sample 270 is accessible from the front by media 280. An opening in the sample holder 240 allows access to the sample 270.

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

The projection 290 may be in the form of an elongated slot 295, as shown in fig. 4. This aspect of the invention allows multiple ones of the samples 270 to be positioned along the trough and imaged using the same microscope module 300. Such an arrangement would allow for high throughput imaging of multiple samples 270.

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

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

As seen in fig. 4, the elongated slot 295 may be moved such that the detection objective 220 and the illumination objective 210 scan the elongated slot 295 to image different ones of the plurality of samples 270. The detection objective 220 and the illumination objective 210 remain fixed to the optical platform.

The media 280 is left undisturbed by either the detection objective or the illumination objective, and remains sterile to allow for long-term experiments.

Reference numerals

10 device

20 laser

25 illumination objective

27 reflection field

30 light sheet

35 illumination beam path

40 samples

45 rotating shaft

50 detector

55 detecting the direction

60 imaging device

65 detection objective lens

66 optical device

67 reflective mirror

70 processor

80 memory storage

85 image

200 microscope device

210 illumination objective

215 illumination beam path

220 detection objective lens

225 detection path

230 immersion medium

240 sample holder

250 wall

260 bottom part

270 sample

280 medium

290 projection

295 groove

300 microscope module

The attached drawing of the abstract is figure 3

FIG. 1 Prior Art

Claims (13)

1. A microscope module (300) for a microscope device (200) for imaging one or more samples (40, 270), the one or more samples (40, 270) being located in a culture medium (280), the microscope device (200) comprising

-a light source (20), the light source (20) for generating an illumination beam along an illumination beam path (215), the illumination beam path (215) being arranged to illuminate a lower surface, including a bottom surface and a side surface, of the one or more samples (40, 270); and

a detector (50), the detector (50) for detecting the emitted light along a detection beam path,

wherein the microscope module (300) comprises:

-a sample holder (240), said sample holder (240) being removably arranged in said microscope device (200), being adapted to hold said one or more samples (40, 270) in a culture medium (280), and having a bottom (260), said bottom (260) being at least partially transparent to said illumination light beam and to emission light emitted from said one or more samples (40, 270);

-at least one illumination objective (210) arranged in the microscope device (200) to direct the illumination beam through an at least partially transparent bottom (260) onto a lower surface, including a bottom surface and side surfaces, of the one or more samples (40, 270);

-at least one detection objective (220) arranged in the microscope device (200) to collect emission light emitted from the one or more samples (270) through the at least partially transparent bottom (260) along a detection path (225);

wherein the detection path (225) is at an angle to the illumination beam path (215);

the illumination objective (210) and the detection objective (220) are located in an immersion medium (230), the immersion medium (230) being in contact with an at least partially transparent bottom (260) of the sample holder (240), and

the immersion medium (230) and the at least partially transparent bottom (260) of the sample holder (240) have substantially the same refractive index to reduce optical aberrations.

2. The microscope module (300) of claim 1, wherein the angle of the detection path (225) to the illumination beam path (215) is substantially a right angle.

3. The microscope module (300) according to claim 1, wherein the at least partially transparent bottom (260) of the sample holder (240) is made of a film attached to a wall (250) of the sample holder (240).

4. The microscope module (300) of claim 1, wherein the at least partially transparent bottom (260) of the sample holder (240) comprises a protrusion (290) adapted to hold the sample (270).

5. The microscope module (300) of claim 4, wherein the illumination beam is arranged to direct the illumination beam through the protrusion (290).

6. The microscope module (300) of claim 4 or 5, wherein the projection (290) is elongate.

7. The microscope module (300) according to any one of claims 1 to 5, wherein the illumination beam path is arranged at 30 ° to a horizontal position.

8. The microscope module (300) according to any one of claims 1 to 5, wherein the refractive index of the culture medium (280) is substantially the same as the refractive index of the at least partially transparent bottom (260) of the sample holder (240).

9. A method of imaging one or more samples (40, 270), comprising:

-placing one or more samples (40, 270) in a sample holder (240), said sample holder (240) having an at least partially transparent bottom (260) and being adapted to hold said one or more samples (40, 270) in a culture medium (280);

-removably arranging the sample holder (240) such that the at least partially transparent bottom (260) contacts an immersion medium (230), the immersion medium (230) having therein an illumination objective (210) and a detection objective (220);

-generating an illumination beam along an illumination beam path (215) by passing light from a light source (20) through the illumination objective (210);

-illuminating a lower surface, including a bottom surface and side surfaces, of the one or more samples (40, 270) through the at least partially transparent bottom (260) with the illumination beam;

-detecting light emitted from the one or more samples (40, 270) through the at least partially transparent bottom (260) along a detection path (225) with the detection objective (220); and

-creating an image of the one or more samples (40, 270);

wherein the detection path (225) is at an angle to the illumination beam path (215); and

wherein the immersion medium (230) and the at least partially transparent bottom (260) of the sample holder (240) have substantially the same refractive index to reduce optical aberrations.

10. The method of claim 9, further comprising selecting different samples from more than one sample (270).

11. The method of claim 9 or 10, wherein the placing comprises placing the one or more samples (40, 270) in a protrusion (290) of an at least partially transparent bottom (260) of the sample holder (240).

12. The method of claim 11, wherein said illuminating further comprises illuminating said one or more samples (40, 270) through said projections (290).

13. The method according to claim 9 or 10, wherein the refractive index of the culture medium (280) is substantially the same as the refractive index of the at least partially transparent bottom (260) of the sample holder (240).