Microscope module for a microscope arrangement for imaging a sample

A microscope module (300) for imaging a sample (270) is disclosed. The microscope module (300) includes at least one illumination objective (210) for producing an illumination beam along an illumination beam path (215) arranged to illuminate lower surface of the sample (270) and at least one detection objective (220) having a detection path (225). The detection path (225) is at an angle to the illumination beam path (215).

CROSS-REFERENCE TO RELATED APPLICATIONS

FIELD OF THE INVENTION

The field of the invention relates to a microscope module for imaging a sample.

BACKGROUND OF THE INVENTION

Selective Plane Illumination Microscopy (SPIM) is a technology that employs generation of a light sheet to illuminate a sample and a perpendicular detection system to enable imaging of optical sections of the samples, which can be living or not. In most embodiments, the SPIM system requires extensive sample preparation to hold the sample in a correct position for imaging. For example, the sample is typically embedded in an agarose cylinder which is submerged in a small chamber filled with an immersion medium, such as water. The technique has been known for over a hundred years, but has only recently found extensive application in imaging biological samples. One disadvantage with the technique is that agarose is not compatible with all biological specimens. The samples are also embedded in vertical cylinders of agarose of limited height in current SPIM systems. This arrangement does not allow for access to the sample during imaging or re-positioning of the sample. The arrangement limits the number of samples that can be imaged since, for example, it is not possible to stack 50 samples in 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 (light sheet) illuminates a sample (specimen) and the sample is viewed through a detector. The axis of the detector is situated substantially perpendicular to the direction of an illumination beam. The sample is displaced through the strip of light and the detector records diffused light from the sample or fluorescent light from the sample in a series of images. Three-dimensional images of the sample can be created by the optical sectioning of the sample and then reconstructing the entire image of the sample.

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

SUMMARY OF THE INVENTION

A microscope module for imaging one or more samples is disclosed. The microscope module comprises an illumination device for producing 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 lower surfaces of one or more of the samples. The illumination beam path is arranged at an angle to the detection path. In one aspect of the disclosure, the angle is substantially orthogonal. The samples are placed in a culture medium. There is no need to mount the samples in a solid or viscous mounting media which might be incompatible with the survival of biological samples and also complicates retrieval and manipulation of the samples.

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. One example of such transparent bottoms is a membrane. The sample holder comprises at least one protrusion in which the sample is held. In one aspect of the disclosure, the protrusion may be in the form of an elongated trough in which a plurality of the samples are held in a culture medium.

The sample holder is arranged to enable easy removal from the microscope module. This enables the samples to be cultured in the sample holder outside of the microscope module and then placed undisturbed into the microscope module for imaging.

The arrangement of this disclosure enables the illumination objective and the detection objective to be placed in an immersion medium that is separate from the culture medium in which the samples are placed. The separation of the culture medium from the immersion medium helps to maintain sterility and also enables the use of small volumes of culture media. The transparent bottom, the immersion medium and the culture medium have substantially the same refractive index to minimise optical aberrations.

The disclosure also teaches a method of imaging a plurality of samples that comprises arranging an illumination objective to illuminate lower surfaces of the plurality of the samples and arranging a detection objective to detect light emitted from the plurality of samples at an approximately orthogonal angle to the illumination beam path. The detected light can be used to create an image of one or more of the plurality of samples.

DETAILED DESCRIPTION OF THE INVENTION

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

FIG. 1illustrates the fundamental principles of SPIM and described more extensively in U.S. Pat. No. 7,554,725, the disclosure of which is incorporated by reference. The arrangement10comprises a laser20, which generates, through an illumination objective25, a light sheet30to illuminate sections of a sample40. The light sheet30is directed along an illumination beam path35. A detection objective65is arranged such that the detection direction55is substantially orthogonal to the plane of the light sheet30(i.e. perpendicular to the illumination beam path35).

The sample40can be rotated about a rotation axis45and the light sheet30can be arranged to illuminate optical sections of the sample40. The laser20typically excites fluorophores in the sample40to emit fluorescent light in many directions.

The detector50detects, through an detection objective65and optical arrangement66, a portion of the emitted fluorescent light from the fluorophores in the sample40that have been excited by the radiation in the light sheet30. The detector50has an imaging device60, such as a CCD camera, that is connected to a processor70with a memory store80. The memory store80stores the individual images85from each of the optical sections of the sample40and the processor70can create a three dimensional image of the sample40.

FIG. 2shows an embodiment of the microscope arrangement200used in this disclosure. Identical reference numerals are used to indicate identical elements inFIGS. 1 and 2. There is no need to embed the sample40in agarose in this disclosure, since the sample40is held sufficiently stable in the apparatus, as will be explained below.

The laser20generates through mirrors67and illumination objective25a light sheet30to illuminate sections of sample40. The light sheet30enters the sample40through the lower surface of the sample40. A large portion of the emitted fluorescent light from the sample40is passed through a detection objective65, reflected by a mirror27and through the optical arrangement66focussed onto the imaging device60in the detector50to form an image. The image from the detector50is passed to the processor70and then stored in the memory store80as individual images85.

FIG. 3shows an example of the microscope module300with an illumination objective210and a detection objective220. The illumination objective210illuminates by an illumination beam (light sheet) along an illumination beam path215. The illumination beam path215through the illumination objective210and a detection path225through the detection objective220are arranged approximately orthogonal to each other. Both the illumination objective210and the detection objective220are located in an immersion medium230, which comprises typically degassed water or immersion oil. Degassing of the water ensures that bubbles are not present in the immersion medium230.

The illumination beam path215through the illumination objective210is located beneath a sample holder240at approximately 30° to the plane of the sample holder240. The detection path225is therefore located at approximately 60° to the plane of the sample holder240. Flexible plastic rings around the illumination objective210and the detection objective220prevent leakage of the immersion medium230.

The sample holder240with walls250is made of a biocompatible material, such as but not limited to PEEK, and has a bottom260that is made of a thin transparent membrane, such as a Teflon® FEP film manufactured by Dupont, having a refractive index substantially similar to that of the immersion medium230and/or the culture medium280to reduce optical aberrations. The transparent membrane in the bottom260allows therefore the passage of radiation onto a sample270located on the top side of the transparent membrane260. The transparent membrane forming the bottom260is attached to the walls250of the sample holder240by biocompatible silicone glue or by clamping. The transparent membrane is curved in the area not supported by the walls250to keep the transparent membrane under tension. The sample holder240is open at the top and the opening enables easy access to and removal of the sample270, if required. The transparent membrane is plasma treated to make it hydrophilic and thus helps to prevent bubble formation in the immersion medium230.

The sample270is located in the curved area in the transparent membrane in a suitable culture medium280. The culture medium280is an embryo or tissue culture medium and may have a layer of oil on its surface to prevent evaporation. The different refractive index of the oil will not affect the imaging of the sample270because the illumination beam path215and/or the detection path225do not pass through the oil. The culture medium280may have a very small volume, for example 10 μl. Examples of such culture media280include, but are not limited to, KSOM, M16 (mouse embryo), DMEM and RPE (cell culture). There is no need to embed the sample270in an agarose cylinder (as known in the art). The protrusion290can be elongated to form a trough (seeFIG. 4).

The microscope module300shown inFIG. 3enables the isolation of the immersion medium230from the culture medium280. It can be seen that this is different than the arrangement10ofFIG. 1in which the immersion medium is the same as the aqueous medium holding the sample40.

The sample270can also be easily manipulated as the sample270is accessible from the top side through the culture medium280. An opening in the sample holder240allows access to the sample270.

It will be seen from the arrangement ofFIG. 3that only the lower surfaces, including bottom surface and side surfaces, of the sample270will be illuminated by the radiation from the illumination objective210. Similarly the fluorescent light from the lower surfaces of the sample270will be collected by the detection objective220and thus used to create the image85in the memory store80.

The protrusion290can be in the form of an elongated trough295, as shown inFIG. 4. This aspect of the invention allows multiple ones of the samples270to be placed along the trough and imaged using the same microscope module300. Such an arrangement will allow high throughput imaging of a plurality of the samples270.

The microscope module300enables long-term high-throughput live cell and embryo imaging experiments, for example, of mammalian embryos and oocytes imaged in vitro.

A method for carrying out long-term high-throughput live cell and embryo imaging experiments can be carried out by the microscope module300. The method comprises arranging the illumination objective210such that an illumination beam is produced to illuminate the lower surfaces of the plurality of samples270along the illumination beam path215. The detection objective220collects a portion the fluorescent light that is emitted from the plurality of samples270. The fluorescent light is emitted in all directions and fluorescent light in an arc of approx. 120° about the detection path225will be collected. The fluorescent light collected by the detection objective220is reflected by a mirror27and through the optical arrangement66focussed onto the imaging device60in the detector50. The imaging device60sends to the processor70data relating to the images85and the processor70is able to create a three-dimensional image of one or more of the plurality of samples270.

It will be seen fromFIG. 4that the elongated trough295can be moved so that the detection objective220and the illumination objective210scan the elongated trough295to image different ones of the plurality of the samples270. The detection objective220and the illumination objective210remain fixed to an optical table.

The culture medium280remains undisturbed by either of the detection objective or of the illumination objective and remains sterile allowing long-term experiments.

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