WO2021147198A1

WO2021147198A1 - Multi-photon microscope transparent specimen adapter and imaging method thereof

Info

Publication number: WO2021147198A1
Application number: PCT/CN2020/087188
Authority: WO
Inventors: 吴科锋; 赖子嘉; 吴瀚; 陈海琪; 叶华; 周心; 朱宇珍; 韦俊任; 戚怡; 林灵辉; 莫小敏
Original assignee: 广东湛江海洋医药研究院; 南方海洋科学与工程广东省实验室(湛江)
Priority date: 2020-01-21
Filing date: 2020-04-27
Publication date: 2021-07-29

Abstract

A multi-photon microscope transparent specimen adapter and an imaging method thereof, the multi-photon microscope transparent specimen adapter comprising a base (1) and a viewing dish (2), the base (1) having disposed thereon an accommodation cavity (111) used to place a biological specimen, and the viewing dish (2) comprising a main body (21), as well as a viewing hole (23) and a connecting sleeve (22) disposed on the main body (21); one end of the connecting sleeve (22) is in communication with the viewing hole (23), and another end is in communication with the accommodation cavity (111). The present invention facilitates fixing and finding the biological specimen, and improves imaging results.

Description

Transparency specimen adapter for multiphoton microscope and imaging method thereof

Technical field

The application belongs to the technical field of experimental equipment, and specifically relates to a transparent specimen adapter for a multiphoton microscope and an imaging method thereof.

Background technique

As we all know, with the emergence of tissue optical transparency technology, the use of multi-photon microscopy (MPM) to obtain mesoscopic systems (such as complete embryos, in vitro organs, and living model animals) on the order of millimeters to centimeters High-precision three-dimensional structure is the current trend and trend of the development of medical molecular diagnostics.

When modern multiphoton microscopes perform ultra-depth-of-field imaging, they generally need to transparentize biological specimens and place them in a petri dish or beaker with a clear solution of a specific refractive index. The microscope objective must be fully immersed in the clear solution during observation. Achieve deep imaging of specimens above the millimeter level. Conventional petri dishes or beakers are often too shallow, too narrow, too high or too wide, which not only makes biological specimens easy to float in the container, insufficient working distance between the lens and other factors that are not conducive to imaging, but also makes the specimen search It becomes more difficult. In addition, the way that biological specimens are glued to containers (petro dishes or beakers) with biological glue, agarose or gelatin, often because the contact surface with the clearing agent is too large, the refractive index of the specimen or the clearing agent is changed. This will affect the transparency and imaging effects of biological specimens.

Therefore, how to effectively solve the problems of difficult fixation, long search time, and poor imaging effect of biological specimens that are transparent in conventional containers is an urgent technical problem to be solved by those skilled in the art.

Summary of the invention

In order to overcome the above-mentioned shortcomings of the prior art, the purpose of this application is to provide a transparent specimen adapter for multiphoton microscopes, which aims to solve the problem of difficult fixation, long search time, and imaging effect of biological specimens that are transparent in conventional containers in the prior art. Poor technical issues.

In order to achieve its purpose, the technical solutions adopted by this application are as follows:

A transparent specimen adapter for a multiphoton microscope, used in conjunction with a multiphoton microscope to obtain a microscopic image of a biological specimen, the multiphoton microscope includes an objective lens for imaging, and is characterized in that the multiphoton microscope is transparent The specimen adapter includes a base and an observation dish matched with the objective lens. The base is provided with a containing cavity for placing the biological specimen. The observation dish includes a body, an observation hole and a connecting sleeve provided on the body Barrel, wherein the first end of the connecting sleeve is communicated with the observation hole, and the second end of the connecting sleeve is communicated with the containing cavity.

Further, the aforementioned transparent specimen adapter for a multiphoton microscope further includes a transparent sleeve, and the second end of the connecting sleeve is communicated with the containing cavity through the transparent sleeve.

Further, one end of the transparent sleeve is threadedly connected with the second end of the connecting sleeve, and the other end can be inserted into the containing cavity.

Further, an outer thread is provided on the outer wall of the connecting sleeve, an inner thread is provided on the inner wall of the receiving cavity, and the connecting sleeve is threadedly connected with the base.

Further, the base is also provided with a plurality of screw mounting holes arranged circumferentially around the accommodating cavity, and each of the screw mounting holes penetrates the inner wall of the accommodating cavity transversely to communicate with the accommodating cavity, wherein Each of the screw mounting holes is equipped with an adjusting screw that can be used to fix the biological specimen placed in the containing cavity.

Further, the base includes a chassis and a transparent cylinder provided on the chassis. The transparent cylinder has the accommodating cavity and has a plurality of screw mounting holes arranged circumferentially around the accommodating cavity. , Wherein the first end of the transparent cylinder is connected with the chassis, and the second end of the connecting sleeve is matched with the second end of the transparent cylinder to communicate with the containing cavity.

Further, the second end of the connecting sleeve is threadedly connected with the second end of the transparent cylinder.

Further, the base is a circular plate.

Further, the observation vessel has a funnel-shaped structure, wherein the inclination angle of the bottom of the main body is 2-5°.

Further, the accommodating cavity is arranged in the middle.

Further, the accommodating cavity is a circular blind hole.

Further, one end of the adjusting screw rod close to the accommodating cavity is a flat end.

Further, one end of the adjusting screw close to the containing cavity is made of a transparent fluorescent material.

Further, the end of the adjusting screw far away from the containing cavity is prismatic.

Correspondingly, this application also proposes an imaging method of the aforementioned transparent specimen adapter for a multiphoton microscope, which includes the following steps:

Injecting an appropriate amount of gel into the containing cavity and placing the processed biological specimen in the gel;

Performing antibody incubation and fluorescent labeling treatment on the biological specimen in the gel;

Installing the observation dish on the base and injecting a clearing agent matching the refractive index of the biological specimen into the containing cavity;

The biological specimen is moved directly below the objective lens of the multiphoton microscope, and the scanning wavelength is set to perform a Z-axis layer scan to obtain an optical image.

Compared with the prior art, the beneficial effects of this application are:

The transparent specimen adapter for the two-photon microscope proposed in the present application provides a accommodating cavity for placing biological specimens on the base, so as to facilitate fixing the biological specimens after injecting a small amount of gel, glue, etc. into the accommodating cavity, and adding transparency The chemical reagents are stored and observed, and the observation dish is detachably connected to the base, so that it is convenient to replace the observation dish of different specifications according to the specifications of the objective lens, so as to facilitate the detection of the sampled specimen. Compared with the prior art, the present application can not only improve the imaging depth and imaging quality of biological specimens, but also shorten the search time for specimens, and at the same time can save the amount of clearing reagents.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present application more clearly, the following will briefly introduce the drawings used in the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present application. Ordinary technicians can obtain other drawings based on these drawings without creative work.

FIG. 1 is a schematic diagram of the overall structure of a transparent specimen adapter for a multiphoton microscope in an embodiment of the application;

FIG. 2 is an exploded schematic diagram of the structure of a transparent specimen adapter for a multiphoton microscope in an embodiment of the application;

3 is a cross-sectional view of the overall structure of a transparent specimen adapter for a multiphoton microscope in an embodiment of the application;

4 is a cross-sectional view of the overall structure of a transparent specimen adapter for a multiphoton microscope in another embodiment of the application;

5 is a cross-sectional view of the overall structure of a transparent specimen adapter for a multiphoton microscope in another embodiment of the application;

6 is a schematic diagram of the overall structure of a transparent specimen adapter for a multiphoton microscope in another embodiment of the application;

FIG. 7 is a top view of the transparent specimen adapter of the multiphoton microscope in an embodiment of the application with the observation glass removed; FIG.

8 is a cross-sectional view of the overall structure of a transparent specimen adapter for a multiphoton microscope in another embodiment of the application;

Figure 9 is a cross-sectional view of an observation vessel in an embodiment of the application;

Fig. 10 is a schematic structural diagram of an adjusting screw in an embodiment of the application.

Description of reference signs:

1-base, 11-transparent cylinder, 111-accommodating cavity, 12-chassis, 2-observation dish, 21-body, 22-connecting sleeve, 23-observation hole, 3-transparent sleeve, 4-adjusting screw.

Detailed ways

In order to be able to understand the above objectives, features and advantages of the application more clearly, the application will be described in detail below with reference to the accompanying drawings and specific implementations. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments can be combined with each other. In the following description, many specific details are set forth in order to fully understand the application, and the described implementations are only a part of the implementations of the application, rather than all of the implementations. Based on the implementation manners in this application, all other implementation manners obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of this application. The terminology used in the specification of the application herein is only for the purpose of describing specific embodiments, and is not intended to limit the application.

1 to 3, an embodiment of the present application provides a transparent specimen adapter for a multiphoton microscope, which is used in conjunction with a multiphoton microscope to obtain microscopic images of biological specimens. The multiphoton microscope includes an objective lens for imaging. The photon microscope transparent specimen adapter includes a base and an observation dish matched with the objective lens. The base is provided with a containing cavity for placing biological specimens. The observation dish includes a body, an observation hole and a connecting sleeve arranged on the body, wherein the connecting sleeve The first end of the barrel is communicated with the observation hole, and the second end of the connecting sleeve is communicated with the containing cavity.

In this embodiment, the above-mentioned multiphoton microscope may be a two-photon microscope, a three-photon microscope, or other types of photon microscopes. The base 1 and the observation bowl 2 are preferably in the shape of a disc, of course, the base 1 can also be rectangular or other shapes. Wherein, the top surface of the base 1 is provided with a containing cavity 111 that can accommodate specimens. The size of the containing cavity 111 can be determined according to the actual situation (that is, the type or size of the specimen), and the observation dish 2 needs to be in accordance with the corresponding photon microscope objective lens. It is set so that the objective lens can observe the specimen in the containing cavity 111 through the observation bowl 2. The accommodating cavity 111 accommodates the specimen, which is helpful to solve the problem that the specimen is easily deformed in the accommodating cavity 111 after using scale A2, SeeDB and other partially transparent reagents, which affects the imaging depth of the photon microscope and the deviation of the imaging position. With a long working distance of 8mm, the commercial objective field of a multiphoton microscope provides multi-dimensional optical deep imaging of the whole biopsy specimen with needle aspiration. At this time, it is preferable that the observation vessel 2 includes a main body 21 and a connecting sleeve 22. The main body 21 can be in the form of a petri dish in an existing laboratory, and the connecting sleeve 22 is located at the bottom of the main body 21, and the main body 21 is also provided with The observation hole 23 communicated with the connecting sleeve 22, and the observation hole 23 can be communicated with the containing cavity 111 through the connecting sleeve 22, so that the objective lens can observe the specimen in the containing cavity 111 through the observation hole 23. As for the connection between the connecting sleeve 22 and the base 1, it may be directly connected, or may be connected to the base 1 through an intermediate component.

When the two-photon microscope transparent specimen adapter in this embodiment is specifically used, the observation dish 2 is removed from the base 1, and a gel with a height of 2 to 3 mm is injected into the accommodating cavity 111, and it is fixed by 4% paraformaldehyde. The puncture sampling specimen is inserted into the gel, so that the biological specimen can be suspended in the accommodating cavity 111. After antibody incubation and fluorescent labeling are carried out according to the experimental procedure, the corresponding clearing reagent is added according to the optical refractive index of different specimens. That is, the clearing reagent can be used to transparentize the biological specimen in all directions, thereby increasing the transparency effect, thereby improving the imaging effect of the biological specimen. When imaging the multiphoton microscope, install the observation dish 2 of the corresponding specification on the base 1, inject the corresponding clearing reagent, move the objective lens of the multiphoton microscope directly above the specimen, set the scanning wavelength, and perform the Z-axis layer scan. It can realize 3D/4D optical imaging with super depth of field.

In this embodiment, by providing the accommodating cavity 111 for placing biological specimens on the base 1, it is not only convenient to fix the biological specimens after the gel is injected into the accommodating cavity 111, but also to place the biological specimens in this way. Compared with the design of the observation chamber in a smaller observation chamber than the conventional container, it not only shortens the search time of the specimen, but also reduces the amount of clearing reagent. At the same time, the observation dish 2 is detachably connected to the base 1, so as to facilitate the replacement according to the specifications of the objective lens. Observation dishes 2 of different specifications, so as to facilitate the detection of sampled specimens, and help to improve the imaging depth and accuracy of the imaging part of the photon microscope.

Further, referring to FIGS. 1 and 2, in an exemplary embodiment, in order to facilitate placing the sampling specimen in the containing cavity 111, the aforementioned transparent specimen adapter for multiphoton microscope further includes a transparent sleeve, wherein the transparent sleeve One end of the barrel 3 can be installed in the accommodating cavity 111, and the other end can be connected to the connecting sleeve 22, so that the second end of the connecting sleeve can communicate with the accommodating cavity through a transparent sleeve. At this time, the length of the connecting sleeve 22 is preferably 2-7 mm, and the inner diameter of the transparent sleeve 3 is preferably 1.8-2.6 mm, the outer diameter is preferably 2.5-4.8 mm, and the length is preferably 4.2-8.4 mm.

When the transparent specimen adapter of the multiphoton microscope in this embodiment is used, the transparent sleeve 3 is installed in the containing cavity 111, and a gel with a height of 2 to 3 mm is injected into the containing cavity 111 through the transparent sleeve 3. It is helpful to observe the gel injection condition (that is, the amount of gel) through the transparent sleeve 3, then remove the transparent sleeve 3, and insert the puncture sample fixed with 4% paraformaldehyde into the gel, and finally Install the transparent sleeve 3 in the accommodating cavity 111 again, and then carry out antibody incubation and fluorescent labeling according to the experimental procedures, and add corresponding clearing reagents according to the optical refractive index of different specimens for storage for later use. When imaging by the multiphoton microscope, connect the observation dish 2 with the transparent sleeve 3, and inject the corresponding clearing reagent, move the objective lens of the multiphoton microscope directly above the specimen, set the scanning wavelength, and perform the Z-axis layer scan. It can realize 3D/4D optical imaging with super depth of field.

Further, referring to FIGS. 1, 2 and 4, in an exemplary embodiment, in order to facilitate the connection of the transparent sleeve 3 with the connecting sleeve 22 in the base 1 and the observation vessel 2, the transparent sleeve 3 is preferably One end of the transparent sleeve 3 is inserted into the containing cavity 111, and the outer wall of the transparent sleeve 3 can be attached to the inner wall of the containing cavity 111, so as to ensure the stability of the transparent sleeve 3 when it is located in the containing cavity 111, and the transparent sleeve 3 The connection with the connecting sleeve 22 can be a threaded connection, that is, an internal thread is provided on the inner wall of the transparent sleeve 3, and an external thread is provided on the outer wall of the connecting sleeve 22, so that the observation vessel 2 can be connected by screwing. Set on the base 1.

Further, referring to FIGS. 1 and 3, in another exemplary embodiment, in order to facilitate the direct connection of the connecting sleeve 22 with the base 1, at this time, it is preferable that the outer wall of the connecting sleeve 22 is provided with an external thread, and the receiving cavity An internal thread is provided on the inner wall of 111, so that the observation vessel 2 is fixed on the base 1 through threaded fit, and at the same time, the observation vessel 2 can be easily disassembled and assembled. At this time, the base 1 is a circular plate, the diameter of the circular plate can be 40mm, the thickness can be 8mm, and the sides have 45 degree chamfers; the depth of the containing cavity 111 is 4mm, the diameter is 10mm; the body 21 is one end An open-ended cylinder, the cylinder has an outer diameter of 44mm, an inner diameter of 40mm, and a length of 12mm; the inner diameter of the observation hole 23 is 6.5mm; the outer diameter of the connecting sleeve 22 is 10mm, the inner diameter is 6.5mm, and the length The length of the external thread on the connecting sleeve 22 is 4 mm.

Further, referring to Figures 3 and 5, as an improvement, in a preferred embodiment, the base 1 is also provided with a plurality of screw mounting holes (not shown in the figure) arranged circumferentially around the containing cavity 111, and Each screw mounting hole transversely penetrates the inner wall of the containing cavity 111 to communicate with the containing cavity 111, wherein each screw mounting hole is installed with an adjusting screw 4 that can be used to fix the biological specimen placed in the containing cavity 111. At this time, the observation vessel 2 can be fixed on the base 1 by means of screw fitting, and when the observation vessel 2 is installed on the base 1, one end of each adjusting screw 4 extending into the containing cavity 111 is located at the lower end of the connecting sleeve 22 and the containing The gap between the bottom of the cavity 111.

When the transparent specimen adapter of the multiphoton microscope in this embodiment is specifically used, the observation dish 2 is removed from the base 1, and then a biological specimen (for example, an irregular bone specimen) is placed in the containing cavity 121, and passed Tighten the adjusting screw 4 to fix the specimen in the accommodating cavity 121, and then carry out antibody incubation and fluorescent labeling according to the experimental procedures, and add the corresponding clearing reagent according to the optical refractive index of different specimens. In this way, the clearing reagent can be used to Biological samples are transparentized in all directions, thereby increasing the transparency effect, thereby improving the imaging effect of biological specimens. When the photon microscope is imaging, install the observation dish 2 of the corresponding specification on the base 1, and inject a clearing reagent matching the refractive index of the current biological specimen through the observation hole 23, and move the objective lens of the multiphoton microscope directly above the specimen. Then adjust the position of the specimen to a proper observation position by observing while using the adjusting screw 4 to adjust the position. After the specimen is fixed in the proper observation position, set the scanning wavelength and perform the Z-axis layer scan, namely It can realize 3D/4D optical imaging with super depth of field.

In this embodiment, by adding a plurality of screw mounting holes arranged circumferentially around the accommodating cavity 111 on the base 1, it is only necessary to install the corresponding adjusting screw 4 in the screw mounting hole during use, and adjust the adjusting screw 4 The depth of extending into the accommodating cavity 121 can fix the transparent biological specimen placed in the accommodating cavity 121 at a position suitable for observation. In this way, the fixation of the specimen is not only convenient, but also can further shorten the time of searching for the specimen under the field of objective lens. At the same time, after the experiment is completed, the adjustment screw 4 only needs to be loosened to take out the biological specimen, so that only one adapter is needed to continuously replace the biological specimen for microscopic scanning, which greatly improves the practicability of the adapter. In addition, because there is no need to add bio-glue, agarose or gel to fix the specimen, it can further avoid the problem that the contact surface between the specimen and the clearing agent is too large, which affects the imaging depth and imaging position deviation of the multiphoton microscope. Improve the imaging effect of biological specimens.

Further, referring to Figures 6 to 8, as an improvement, in another preferred embodiment, the base 1 includes a chassis 12 and a transparent cylinder 11 arranged on the chassis 12, and the transparent cylinder 11 has an accommodating cavity 111 and There are a plurality of screw mounting holes arranged circumferentially around the containing cavity 111, wherein the adjusting screw 4 is installed in each screw mounting hole, the first end of the transparent cylinder 11 is connected with the chassis 12, and the connecting sleeve The second end of 22 is matched with the second end of the transparent cylinder 11 to communicate with the containing cavity 111. Illustratively, the chassis 12 is preferably a circular plate. Of course, the chassis 11 may also be a rectangular plate, a triangular plate or other shapes, as long as the stability of the entire base 1 can be ensured, which is not specifically limited. Illustratively, the transparent cylindrical body and the chassis 12 are an integral structure, so that it can be integrally formed (such as integral injection molding) during manufacturing, which saves manufacturing costs. Illustratively, the number of adjusting screws 4 provided on the transparent cylinder 11 is four, and each adjusting screw 4 is arranged circumferentially around the containing cavity 111 at an interval of 90°. Of course, in some other embodiments, the adjusting screw 4 The number can also be two, three, five, six, etc. (correspondingly, the number of screw mounting holes can also be two, three, five, six, etc.), and the number can be changed according to actual needs Certainly.

In this embodiment, by arranging the accommodating cavity 111 and the adjusting screw 4 on the transparent cylinder 11, during the experiment, it is convenient to observe the injection amount of the clearing reagent and the specimen in the accommodating cavity 111 through the transparent cylinder 11 Experimental status.

Further, referring to FIGS. 6 and 8, in an exemplary embodiment, the second end of the connecting sleeve 22 is threadedly connected with the second end of the transparent cylinder 11. Illustratively, the inner wall of the second end of the connecting sleeve 22 is provided with an internal thread (not shown in the figure), and the outer wall of the second end of the transparent cylinder 11 is provided with an external thread (not shown in the figure), thereby The detachable connection between the observation vessel 2 and the base 1 is realized through the threaded connection mode, which is simple and reliable, and is easy to operate. At this time, more specifically, the outer diameter of the transparent cylinder 11 can be 20-24mm, and the length of the external thread can be 4-6mm; when the chassis 11 is a circular plate, its diameter can be 46-52mm, and the thickness can be Correspondingly, the inner diameter of the connecting sleeve 22 can be 20-24mm, the outer diameter can be 26-30mm, and the length can be 4-6mm; the inner diameter of the observation hole 23 can be 20-24mm; the body 21 has one end An open-ended cylinder, the cylinder may have an inner diameter of 48-52mm, an outer diameter of 54-58mm, and a length of 10-14mm.

Further, referring to FIG. 5, FIG. 6, FIG. 7, FIG. 8 and FIG. 10, in an exemplary embodiment, the end of the adjusting screw 4 close to the accommodating cavity 111 is a flat end. Illustratively, the diameter of the end of the adjusting screw 4 close to the accommodating cavity 111 is smaller than the diameter of the end away from the accommodating cavity 111.

In this embodiment, by designing the end of the adjusting screw 4 close to the accommodating cavity 111 with a flat head, the end of the adjusting screw 4 close to the accommodating cavity 111 can be prevented from being too sharp and damaging the specimen.

Further, referring to FIGS. 5, 6, 7, 8, and 10, in an exemplary embodiment, the end of the adjusting screw 4 close to the accommodating cavity 111 is made of a transparent fluorescent material, so that the transparent fluorescent material is used The emitted luminosity can make it easier to check the position of the specimen in the containing cavity 111, and then it is more convenient to fix the specimen placed in the containing cavity 111 at the position to be observed, which is beneficial to further shorten the search under the objective lens. The time of the specimen.

Further, referring to Figures 5, 6, 7, 8, and 10, in an exemplary embodiment, the end of the adjusting screw 4 away from the accommodating cavity 111 is prismatic. Illustratively, the adjusting screw 4 is away from the housing 111. One end of the cavity 111 is in the shape of a hexagonal prism. At this time, in a more specific embodiment, as shown in FIG. 10, the adjusting screw 4 is mainly composed of a cylindrical portion (not shown in the figure) without threads, a truncated portion (not shown in the figure), and The threaded cylindrical part (not shown in the figure) and a hexagonal prism-shaped prism part (not shown in the figure), wherein the diameter of the cylindrical part without thread (that is, the above-mentioned flat end) can be 0.4 to 0.6 mm , The length can be 0.8～1.2mm, the length of the truncated part can be 3～5mm, the diameter of the threaded cylindrical part can be 1～2mm, the length can be 5～7mm, the length of the hexagonal prism can be 12～16mm.

In this embodiment, since the adjusting screw 4 is relatively small, for ease of use, the end of the adjusting screw 4 away from the accommodating cavity 111 is designed as a prismatic shape. In this way, the shape of the head and the tail of the adjusting screw 4 can be matched (such as the end face). All are regular hexagons) using tools to adjust the length of the adjusting screw 4 extending into the accommodating cavity 111.

Further, referring to the figure, in an exemplary embodiment, in order to facilitate the clearing reagent to enter the containing cavity 111 through the observation hole 23, it is preferable that the observation dish 2 has a funnel-shaped structure, and the inclination angle of the bottom of the body 21 is preferably 2 to 5 degrees, in which the smaller open end of the funnel structure (ie, the observation hole 23) is connected to the connecting sleeve 22, thereby helping to avoid the phenomenon of wasting the clearing agent, thereby saving costs. At this time, in some embodiments, as shown in FIGS. 1 to 4, the body 21 is preferably a disc body, and the height of the body 21 is preferably 10-15 mm, and the diameter is longer than that of Olympus, Leica, Nikon, Zeiss, etc. (WD≥4mm) length 20～80mm (ie 75～110mm), and the inner diameter of the observation hole 23 is preferably 2.51～4.81mm; and in other embodiments, referring to Figures 5 to 9, the body 21 is preferably a disc The height of the main body 21 is preferably 10-14 mm, and the diameter is preferably 54-58 mm.

Further, referring to FIGS. 2 and 7, in some exemplary embodiments, in order to improve the stability of the observation vessel 2 after being installed on the base 1, it is preferable that the receiving cavity 111 is arranged in the center. Illustratively, referring to FIGS. 2 to 5, in some embodiments, the receiving cavity 111 is preferably located at the center of the top surface of the base 1, that is, when the base 1 is a circular plate, the receiving cavity 111 and the base 1 are arranged concentrically. Illustratively, referring to FIGS. 6 to 8, in other embodiments, the transparent cylinder 11 is located at the center of the chassis 12, and the accommodating cavity 111 provided in the transparent cylinder 11 is located at the center of the transparent cylinder 11. The position, that is, when the chassis 12 is a circular plate, the accommodating cavity 111 and the chassis 12 are arranged concentrically.

Further, referring to FIG. 2 and FIG. 7, in some exemplary embodiments, the receiving cavity 111 is a circular blind hole. Illustratively, referring to Figures 2 to 5, in some embodiments, the circular blind hole has a depth of 1.5-4mm and a diameter of 2-10mm, where the best depth is 4mm and the best diameter is It is 6mm, which is beneficial to reduce the clearing reagent while maintaining the accuracy of specimen observation. Illustratively, referring to Figures 6 to 8, in other embodiments, the circular blind hole has a depth of 8-16mm and a diameter of 6-12mm. Among them, the best depth is 12mm, and the best The diameter is set to 10mm, which also helps to reduce the clearing reagent while maintaining the accuracy of the specimen observation.

Correspondingly, referring to FIG. 1 to FIG. 4, an imaging method of the aforementioned transparent specimen adapter for a multiphoton microscope includes the following steps:

Step S1, inject an appropriate amount of gel into the containing cavity 111 and place the processed biological specimen in the gel;

Step S2, performing antibody incubation and fluorescent labeling treatment on the biological specimen in the gel;

Step S3, installing the observation dish 2 on the base 1 and injecting a clearing agent matching the refractive index of the biological specimen into the containing cavity 111;

Step S4, move the biological specimen directly below the objective lens of the multiphoton microscope, and set the scanning wavelength to perform a Z-axis slice scan to obtain an optical image.

In the above step S1, the method of injecting the gel into the accommodating cavity 111 can be a corresponding syringe or other tools, and the type of gel can be an existing appropriate type. Of course, before placing the biological specimen in the gel, it also includes pre-processing the specimen, such as fixing the biological specimen with 4% paraformaldehyde.

In the above step S2, the antibody incubation conditions (such as dilution and temperature) are set according to the actual biological specimen to meet the optimal conditions of the biological specimen to be tested. The dye for fluorescent labeling is set according to the actual situation, such as the type of biological sample and the color to be imaged.

In the above step S3, by installing the observation dish 2 of the corresponding specification on the base 1, and injecting the corresponding clearing reagent into the accommodating cavity 111 from the observation hole 23 on the observation dish 2, the biological specimen can be set at a preset time. Afterwards, it is in a transparent state to facilitate the observation of biological specimens.

In the above step S4, by placing the transparent specimen adapter of the multiphoton microscope on the stage of the multiphoton microscope and placing the biological specimen in the containing cavity 111 directly below the objective lens, the relevant setting can be set on the multiphoton microscope. The corresponding optical image can be obtained after the parameters of, and the corresponding operation steps can refer to the existing method of using the multiphoton microscope, which will not be described in detail here.

Specifically, when using the multiphoton microscope transparent specimen adapter in this application: the diameter of the special lens for the transparent biological specimen of the conventional multiphoton microscope is 75-100mm, the test OLYMPUS FMPE-RS, the objective lens XLSLPLN25XSVMP2, the working distance is 8mm, the magnification is 25X, and the refraction Rate: 1.33～1.42.

Example: Transparency specimen adapter for multiphoton microscope (the size of the body 2 is within the range of the marked size, and the tilt angle of the bottom of the body 2 is 5°C)

Comparative example 1: Transparency specimen adapter for multiphoton microscope (non-standard), disk-shaped top, opening >110mm, taking diameter 150mm as an example;

Comparative example 2: Transparency specimen adapter for multiphoton microscope (non-standard), disc-shaped top, flat bottom

Comparative Example 1: Standard Petri Dish (100mm diameter, 21mm height)

Comparative example 2: 300ml beaker (85mm diameter, 108mm height)

Object: A 10mm section of mouse hemibrain treated with cubic clearing reagent, labeled with DEXTRAN-FITC, and imaged.

Table 1

As shown in Table 1, compared with conventional containers (such as petri dishes, beakers, etc.) for placing biological specimens, the multiphoton microscope transparent specimen adapter in this application can achieve the goal with a smaller amount of transparentizing reagents. The fastest and best imaging effect.

It should be noted that other contents of the transparent specimen adapter and imaging method of the multiphoton microscope disclosed in the present application can be referred to the prior art, and will not be repeated here.

The above are only preferred embodiments of the present application, and do not impose any formal restrictions on the present application. Therefore, any modifications or equivalent changes made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application And modifications are still within the scope of the technical solution of this application.

Claims

A transparent specimen adapter for a multiphoton microscope, which is used in conjunction with a multiphoton microscope to obtain microscopic images of biological specimens, the multiphoton microscope includes an objective lens for imaging, and is characterized in that the multiphoton microscope is transparent The specimen adapter includes a base and an observation dish matched with the objective lens. The base is provided with an accommodation cavity for placing the biological specimen. The observation dish includes a body, an observation hole and a connecting sleeve provided on the body Barrel, wherein the first end of the connecting sleeve is communicated with the observation hole, and the second end of the connecting sleeve is communicated with the containing cavity.
The transparent specimen adapter for a multiphoton microscope according to claim 1, further comprising a transparent sleeve, and the second end of the connecting sleeve is communicated with the containing cavity through the transparent sleeve.
The transparent specimen adapter for a multiphoton microscope according to claim 2, wherein one end of the transparent sleeve is threadedly connected with the second end of the connecting sleeve, and the other end can be inserted into the containing cavity .
The transparent specimen adapter for a multiphoton microscope according to claim 1, wherein an outer thread is provided on the outer wall of the connecting sleeve, and an inner thread is provided on the inner wall of the accommodating cavity, and the connecting sleeve is connected with The base is screwed and connected.
The transparent specimen adapter for a multiphoton microscope according to claim 1, wherein the base is further provided with a plurality of screw mounting holes arranged circumferentially around the accommodating cavity, and each of the screw mounting holes penetrates transversely The inner wall of the accommodating cavity is in communication with the accommodating cavity, wherein each screw mounting hole is equipped with an adjusting screw that can be used to fix the biological specimen placed in the accommodating cavity.
The transparent specimen adapter for a multiphoton microscope according to claim 5, wherein the base comprises a chassis and a transparent cylinder provided on the chassis, and the transparent cylinder has the containing cavity and thereon A plurality of the screw mounting holes arranged circumferentially around the accommodating cavity, wherein the first end of the transparent cylinder is connected to the chassis, and the second end of the connecting sleeve is connected to the transparent cylinder The second end is matched to communicate with the containing cavity.
The transparent specimen adapter for a multiphoton microscope according to claim 6, wherein the second end of the connecting sleeve is threadedly connected with the second end of the transparent cylinder.
The transparent specimen adapter for a multiphoton microscope according to any one of claims 1 to 5, wherein the base is a circular plate.
The transparent specimen adapter for a multiphoton microscope according to any one of claims 1 to 7, wherein the observation vessel has a funnel-shaped structure, and the inclination angle of the bottom of the main body is 2-5°.
The transparent specimen adapter for a multiphoton microscope according to any one of claims 1 to 7, wherein the accommodating cavity is centrally arranged.
The transparent specimen adapter for a multiphoton microscope according to any one of claims 1 to 7, wherein the accommodating cavity is a circular blind hole.
The transparent specimen adapter for a multiphoton microscope according to any one of claims 5 to 7, wherein the end of the adjusting screw near the accommodating cavity is a flat end.
The transparent specimen adapter for a multiphoton microscope according to claim 12, wherein the end of the adjusting screw near the accommodating cavity is made of a transparent fluorescent material.
The transparent specimen adapter for a multiphoton microscope according to claim 12, wherein the end of the adjusting screw away from the containing cavity is prismatic.
A method for imaging a transparent specimen adapter for a multiphoton microscope according to any one of claims 1, 2, 3, 4, 8, 9, 10, 11, characterized in that it comprises the following steps:

Injecting an appropriate amount of gel into the containing cavity and placing the processed biological specimen in the gel;

Performing antibody incubation and fluorescent labeling treatment on the biological specimen in the gel;

Installing the observation dish on the base and injecting a clearing agent matching the refractive index of the biological specimen into the containing cavity;

The biological specimen is moved directly below the objective lens of the multiphoton microscope, and the scanning wavelength is set to perform a Z-axis layer scan to obtain an optical image.