CN212077049U - Coupling device and micro-optical tweezers single cell sorting system - Google Patents

Abstract

The utility model discloses a coupling device and a micro-optical tweezers single cell sorting system. The coupling device comprises a top interface, a filter holder and a dichroic mirror holder, a bottom interface, a first support plate and a second A support plate, the top interface and the bottom interface are placed in parallel, connected by a first support plate and a second support plate, the first support plate is provided with a light hole, and the dichroic mirror frame is mounted on the second support plate , the filter holder is mounted on the first support plate, and the top interface, the filter holder, the dichroic frame and the bottom structure are all provided with concentric circular holes. Through the coupling device, the optical tweezers can be easily and quickly introduced into the commercial microscope, and at the same time, the sorting system is added to constitute a micro-optical tweezers sorting system and method, which breaks the limitation that the commercial microscope is only used for observation. Multiple functions are realized on one system: single cell observation, capture and manipulation, and separation and extraction.

Description

Coupling device and micro-optical tweezers single cell sorting system

technical field

The utility model relates to the field of optical instruments, in particular to a coupling device and a microscopic-optical tweezers single-cell sorting system.

Background technique

Single-cell analysis can reveal the basic unit of life—the diversity and difference of cellular material composition and physiological behavior, and is the mainstream cutting-edge technology in today’s life analysis. Single-cell analysis is the study of individual differences in cell populations, including cell size, growth rate, chemical composition (phospholipids, proteins, metabolites, DNA/RNA), etc., as well as the causes and mechanisms of differences between cells. Its research content involves the fields of tumor biology, stem cells, microbiology, neurology and immunology.

The biggest challenge in single-cell analysis lies in the small size of cells, and the complex chemical composition and difficulty of trace components brought about by the small size. The size of a single cell is mostly distributed on the micron scale or even sub-micron scale, and the manipulation and analysis of a single cell at this scale put forward extremely high requirements on the precision and sensitivity of the instrument. Therefore, to achieve single-cell analysis, the following problems must first be solved: single-cell imaging, single-cell signal acquisition, single-cell capture, separation and extraction.

An optical microscope is an optical instrument that uses optical principles to magnify and image tiny objects that cannot be distinguished by the human eye for extracting microstructural information. It is a sign that mankind has entered the atomic age. Optical microscope is one of the commonly used tools for single cell research and detection. It can observe the morphology and fluorescence characteristics of single cells, and realize the qualitative and quantitative research of single cells.

However, the observation of single cells by optical microscopy is limited to the observation of cell morphology, cell structure, fluorescence specificity, etc., and the manipulation, capture, separation and extraction of single cells cannot be realized, which greatly limits the subsequent operations on the observed specific cells. (such as cell culture, single-cell sequencing, etc.). In addition, as a mature commercial product on the market, the optical microscope has a mature optical path structure and mechanical structure. It is one of the difficulties to introduce the required modules without affecting the performance of the microscope.

Utility model content

Based on the prior art, the purpose of the present invention is to provide a coupling device that enables the introduction of an optical tweezers single cell sorting module on the basis of the existing microscope, so as to realize the simultaneous observation, manipulation and detection of single cells on one device. separation.

One aspect of the present invention provides a coupling device, the coupling device includes a top interface, a filter holder and a dichroic mirror frame, a bottom interface, a first support plate and a second support plate, the top interface and the bottom The interfaces are placed in parallel and are connected by a first support plate and a second support plate, the first support plate is provided with a light hole, the dichroic mirror frame is installed on the second support plate, and the filter frame is installed on the second support plate. On the first support plate, the top interface, the filter holder, the dichroic frame and the bottom structure are all provided with concentric circular holes.

In another preferred embodiment, the filter holder and the dichroic mirror frame are installed by a screw, and the mirror frame can be adjusted by manually or electrically rotating the screw.

In another preferred embodiment, multiple groups of filter frames are arranged in the coupling device.

In another preferred example, the top interface, the bottom interface, the first support plate and the second support plate are an integrated structure or a separate structure.

In another preferred example, threads are provided inside the light-passing holes on the first support plate.

In another preferred example, the bottom interface and the bottom interface include a dovetail shape, a groove shape, and the like.

Another aspect of the present utility model provides a micro-optical tweezers single cell sorting system, comprising:

- Microscope: for single cell signal detection;

-Coupling device: for coupling microscope and optical tweezers capture module;

-Optical tweezers capture module: used to capture and manipulate single cells with the carrier platform;

- Sorting module: for single cell sorting;

-Computer: connect with microscope and optical tweezers capture module respectively and carry out program control.

The microscope is one of a brightfield microscope, a fluorescence microscope or a Raman microscope.

In another preferred embodiment, the optical tweezers capture module includes a laser and a beam shaping device.

In another preferred example, the wavelength of the laser output laser light is one of 532 nm, 785 nm or 1064 nm.

In another preferred example, the wavelength of the laser output from the laser is 1064 nm.

In another preferred embodiment, the beam shaping device is a beam expander collimation component.

In another preferred embodiment, the laser is coaxial with the beam expander and collimator assembly.

In another preferred embodiment, the optical tweezers capture module includes single optical trap optical tweezers or multiple optical trap optical tweezers.

In another preferred embodiment, the multi-optical trap optical tweezers are holographic optical tweezers.

The microscope includes an eyepiece imaging module, a microscope adapter plate, an objective lens microscope module, and a microscope skeleton module. The microscope adapter plate and the objective lens microscope module are installed on the microscope skeleton; the top interface of the coupling device is connected to the eyepiece imaging module. The bottom interface is connected with the microscope adapter board, and the optical tweezers capture module is fixed on the microscope adapter board.

The microscope includes an eyepiece imaging module, a detection and identification module, a microscope adapter plate, an objective microscope module, and a microscope skeleton module. The detection and identification module is connected to the eyepiece imaging module, and the microscope adapter plate and the objective lens microscope module are installed on the On the microscope frame, the top interface of the coupling device is connected to the detection and identification module, the bottom interface is connected to the microscope adapter plate, and the optical tweezers capture module is fixed on the microscope adapter plate.

In another preferred embodiment, the detection and identification module is a fluorescence detection module or a Raman detection module.

In another preferred embodiment, the sorting module includes a microfluidic chip and a sample feeding device, and the microfluidic chip is placed on the microscope skeleton carrier platform.

In another preferred embodiment, the sampling device is a gravity-driven adjustable sampling device, a syringe pump or a peristaltic pump.

In another preferred embodiment, the gravity-driven adjustable sample introduction device includes a height-adjustable sample holder, a sample container, and a conduit, the conduit is connected to the sample container and the microfluidic chip, and the sample container is mounted on the adjustable sample The lifting and lowering of the adjustable sample rack drives the sample in the sample container to inject into the microfluidic chip to realize the microflow of the sample solution.

The beneficial effects of the present utility model are:

(1) Breaking the limitations of the mature optical path structure and mechanical structure of commercial microscopes, without affecting the performance of the microscope, other modules are introduced into the microscope through the simple and compact structure of the coupling device;

(2) By coupling optical tweezers on commercial microscopes and adding single cell sorting function, the limitation that the microscope is only suitable for observation and monitoring is improved, and the capture, manipulation, separation and extraction of single cells are realized.

Description of drawings

In the accompanying drawings, identical parts and features have the same reference numerals. Many of the drawings are schematic and may not be to scale.

Figure 1 is a schematic diagram of the structure of the coupling device;

Figure 2 is a schematic diagram of the structure of the micro-optical tweezers single-cell sorting system;

Figure 3 is a schematic diagram of the optical path of the single optical trap optical tweezers capture module;

Figure 4 is a schematic diagram of the optical path of the holographic optical tweezers capture module;

Figure 5. Schematic diagram of the structure of the brightfield-optical tweezers single-cell sorting system.

The specific reference signs are as follows:

1. Coupling device; 2. Optical tweezers capture module; 3. Gravity-driven adjustment sampling device; 4. Microfluidic chip; 5. Image acquisition device; 6. Eyepiece imaging module; 7. Detection and identification module; 8. Microscope adapter plate; 9. Objective microscope module ;10 Microscope frame; 11 Top interface; 12 Filter holder; 13 First support plate; 14 Bottom interface; 15 Dichroic mirror frame; 16 Second support plate; 17 Laser; Beam of microscope optical path; 20 microscope objective, 211/2 glass; 22 polarizing beamsplitter; 231/4 glass; 24 spatial light modulator; 25 first doublet; 26 reflector; 27 second doublet .

Detailed ways

In order to facilitate the understanding of the embodiments of the present utility model, several specific embodiments will be further explained below with reference to the accompanying drawings, and each embodiment does not constitute a limitation to the embodiments of the present utility model. In addition, the accompanying drawings are schematic diagrams, so the apparatus and apparatus of the present invention are not limited by the size or scale of the schematic diagrams.

It should be noted that, in the claims and description of this patent, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or Any such actual relationship or order between these entities or operations is implied. Moreover, the terms "comprising", "comprising" or any other variation thereof are intended to encompass a non-exclusive inclusion such that a process, method, article or device that includes a list of elements includes not only those elements, but also includes not explicitly listed or other elements inherent to such a process, method, article or apparatus. Without further limitation, an element qualified by the phrase "comprising a" does not preclude the presence of additional identical elements in a process, method, article, or device that includes the element.

Example 1

Figure 1 is a schematic diagram of the structure of the coupling device, which specifically includes:

The top interface 11, the filter holder 12 and the dichroic mirror holder 15, the bottom interface 14, the first support plate 13 and the second support plate 16, the top interface 11 and the bottom interface 14 are placed in parallel, through the first support plate 13 is connected to the second support plate 16, the first support plate 13 is provided with a light-passing hole, the dichroic mirror frame 15 is mounted on the second support plate 16, and the filter holder 12 is mounted on the first support On the board 13 , the top interface 11 , the filter holder 12 , the dichroic frame 15 and the bottom structure 14 are all provided with concentric circular holes.

Preferably, the filter holder 12 and the dichroic mirror frame 15 are installed by a screw, and the mirror frame can be adjusted by adjusting the rotating screw.

Preferably, multiple groups of filter frames are arranged in the coupling device.

Preferably, the top interface 11 , the bottom interface 14 , the first support plate 13 and the second support plate 16 are an integrated structure or a separate structure.

Preferably, threads are provided inside the light-passing holes on the first support plate 13 .

Preferably, the bottom interface and the bottom interface include but are not limited to a dovetail shape, a groove shape, and the like.

The structure of the coupling device 1 for coupling the microscope and the optical tweezers capture module is shown in Figure 2:

The microscope includes an eyepiece imaging module 6, a detection and identification module 7, a microscope adapter plate 8, an objective microscope module 9, and a microscope skeleton module 10. The detection and identification module 7 is connected to the eyepiece imaging module 6, and the microscope adapter plate 8 Installed on the microscope frame 10 with the objective microscope module 9, the top interface of the coupling device 1 is connected with the detection and identification module 7, the bottom interface is connected with the microscope adapter plate 8, and the optical tweezers capture module 2 is fixed on the microscope adapter plate 8; The beam of the original microscope optical path enters the coupling device 1 through the top interface of the coupling device 1, and the direction of the optical path does not change. The beams of the optical paths are spatially coincident, and the two beams enter the objective microscope module 9 at the same time, and single cell observation, capture and manipulation can be performed by adjusting the focusing magnification of the objective microscope module 9 and the microscope stage by computer or manually.

Example 2

As shown in Figure 2, a fluorescence-optical tweezers single-cell sorting system includes:

- fluorescence microscope, - coupling device, - optical tweezers capture module, - sorting module, - computer (not marked in the figure);

Fluorescence microscope, including eyepiece imaging module 6, detection and identification module 7, microscope adapter plate 8, objective microscope module 9, microscope frame 10, described eyepiece imaging module 6, detection and identification module 7, objective microscope module 9, microscope frame 10 is a commercial microscope, which can be disassembled and assembled arbitrarily, the detection and identification module 7 is a fluorescence detection module, the detection and identification module 7 is connected with the eyepiece imaging module 6, and the microscope adapter plate 8 is installed on the objective microscope module 9. On the microscope frame 10, the top interface of the coupling device 1 is connected to the detection and identification module 7, the bottom interface is connected to the microscope adapter plate 8, and the optical tweezers capture module 2 is fixed on the microscope adapter plate; the sorting module includes a microfluidic chip 4 , Gravity drives and adjusts the sample introduction device 3, and the microfluidic chip 4 is placed on the microscope skeleton carrier platform.

The optical tweezers capturing module 2 is a single optical trap optical tweezers, as shown in FIG. 3 : including a laser 17 and a beam shaping device. The beam shaping device is the beam expanding and collimating component 18, and the optical path of the optical tweezers capture module is shown in Figure 3: the original microscope optical path beam 19 enters the coupling device through the top interface of the coupling device, and filters the stray light through the filter, and passes through the two-way The direction of the optical path does not change after the chromatic mirror, and the light beam of the optical tweezers capture module enters the coupling device through the light hole on the first support plate. At the same time, the light beam enters the microscope objective and is focused. At this time, a single optical trap will be formed at the focused position to realize single cell capture.

In addition, the optical tweezers capture module can also be multi-trap optical tweezers, including but not limited to holographic optical tweezers. The specific structure is shown in Figure 4:

The laser 17 emits laser light, and the laser passes through the beam expanding and collimating component 18 to generate parallel light, and the parallel light passes through the 1/2 glass plate 21 and the polarization beam splitter prism 22. The combination of the two can adjust the polarization of the incident light by adjusting the 1/2 wave plate 21. direction, so that the incident light reaches the maximum transmittance after passing through the polarizing beam splitter prism 22, and reaches the spatial light modulator 24 with the maximum utilization rate of light energy. The image is illuminated by the incoming light, returns along the original optical path, passes through the 1/4 glass 23, adjusts the 1/4 glass 23 to make the beam achieve the maximum contrast, and can make the beam passing through the polarizing beam splitter prism 22 achieve the maximum reflection At this time, the computational hologram carrying the multi-optical trap information passes through the first doublet lens 25, the reflecting mirror 26, and the second doublet lens 27 in sequence, and at the dichroic mirror of the coupling device and the beam 19 of the original microscope optical path coincide, and enter the microscope objective lens 20 to focus at the same time, and at this time, multiple optical traps will be formed at the focus position to realize the capture of multiple single cells.

The gravity-driven adjustable sample introduction device 3 includes a height-adjustable sample holder, a sample container, and a conduit, the conduit is connected to the sample container and the microfluidic chip 4, the sample container is mounted on the adjustable sample holder, and the adjustable sample The rise and fall of the rack drives the sample in the sample container to inject into the microfluidic chip 4 to realize the microflow of the sample solution.

Preferably, the sampling device includes, but is not limited to, a gravity-driven regulated sampling device, a syringe pump or a peristaltic pump and other sampling devices.

The specific fluorescence-optical tweezers single cell sorting method includes the following steps:

(1) Place the microfluidic chip on the microscope stage;

(2) inject the cell sample into the sample container, adjust the gravity drive to adjust the sample introduction device, and make the sample enter the microfluidic chip channel;

(3) Obtaining single-cell fluorescence information through the fluorescence detection module, and processing the fluorescence image through a computer to identify the required sample according to the single-cell fluorescence information;

(4) Capture the single cell required by optical tweezers, and use the optical tweezers to move the single cell to the vicinity of the sampling point of the sample droplet;

(5) Push the single cell out of the channel through the sorting module to the droplet sampling point to form a single-cell droplet package;

(6) Use the capillary to wrap the droplets and export them into the capillary for later cultivation, expansion, and the like.

Example 3

As shown in Figure 3, a Raman-optical tweezers single cell sorting system includes:

- Raman microscope, - coupling device, - optical tweezers capture module, - sorting module, - computer (not marked in the figure);

Raman microscope, including eyepiece imaging module 6, detection and identification module 7, microscope adapter plate 8, objective microscope module 9, microscope skeleton 10, described eyepiece imaging module 6, detection and identification module 7, objective microscope module 9, microscope The skeleton 10 is a commercial microscope, which can be disassembled and assembled arbitrarily. The detection and identification module 7 is a Raman detection module. The detection and identification module 7 is connected to the eyepiece imaging module 6. The microscope adapter plate 8 is connected to the objective microscope module 9. Installed on the microscope frame 10, the top interface of the coupling device 1 is connected to the detection and identification module 7, the bottom interface is connected to the microscope adapter plate 8, and the optical tweezers capture module 2 is fixed on the microscope adapter plate; the sorting module includes a microfluidic control board. The chip 4, the gravity-driven adjustment sample feeding device 3, and the microfluidic chip 4 are placed on the microscope skeleton carrier platform.

The optical tweezers capturing module 2 is a single optical trap optical tweezers, as shown in FIG. 3 : including a laser 17 and a beam shaping device. The beam shaping device is the beam expanding and collimating component 18, and the optical path of the optical tweezers capture module is shown in Figure 3: the original microscope optical path beam 19 enters the coupling device through the top interface of the coupling device, and filters the stray light through the filter, and passes through the two-way The direction of the optical path does not change after the chromatic mirror, and the light beam of the optical tweezers capture module enters the coupling device through the light hole on the first support plate. At the same time, the light beam enters the microscope objective and is focused. At this time, a single optical trap will be formed at the focused position to achieve single cell capture.

The gravity-driven adjustable sample introduction device 3 includes a height-adjustable sample holder, a sample container, and a conduit, the conduit is connected to the sample container and the microfluidic chip 4, the sample container is mounted on the adjustable sample holder, and the adjustable sample The lifting and lowering of the rack drives the sample in the sample container to inject into the microfluidic chip 4 to realize the microflow of the sample solution;

The specific Raman-optical tweezers single cell sorting method includes the following steps:

(1) Place the microfluidic chip on the microscope stage;

(2) inject the cell sample into the sample container, adjust the gravity drive to adjust the sample introduction device, and make the sample enter the microfluidic chip channel;

(3) Obtain the single-cell Raman information through the Raman detection module, analyze the obtained Raman spectrum signal by computer or compare it with the standard Raman spectrum signal in the constructed single-cell phenotype database, which is given by the computer Analyze the result to determine whether it is the required single cell;

(4) Capture the single cell required by optical tweezers, and use the optical tweezers to move the single cell to the vicinity of the sampling point of the sample droplet;

(5) Push the single cell out of the channel through the sorting module to the droplet sampling point to form a single-cell droplet package;

(6) Use the capillary to wrap the droplets and export them into the capillary for later cultivation, expansion, and the like.

Example 4

As shown in Figure 2, a brightfield-optical tweezers single-cell sorting system includes:

- brightfield microscope, - coupling device, - optical tweezers capture module, - sorting module, - computer (not marked in the figure);

The Raman microscope includes an eyepiece imaging module 6, a detection and identification module 7, a microscope adapter plate 8, an objective microscope module 9, and a microscope skeleton 10. The eyepiece imaging module 6, the objective microscope module 9, and the microscope skeleton 10 are commercialized The microscope can be disassembled and assembled arbitrarily. The microscope adapter plate 8 and the objective microscope module 9 are installed on the microscope frame 10, the top interface of the coupling device 1 is connected with the eyepiece imaging module 7, and the bottom interface is connected with the microscope adapter plate 8. The optical tweezers capture module 2 is fixed on the microscope adapter plate; the sorting module includes a microfluidic chip 4, a gravity-driven adjustment sample feeding device 3, and the microfluidic chip 4 is placed on the microscope skeleton carrier platform.

The optical tweezers capturing module 2 is a single optical trap optical tweezers, as shown in FIG. 3 : including a laser 17 and a beam shaping device. The beam shaping device is the beam expanding and collimating component 18, and the optical path of the optical tweezers capture module is shown in Figure 3: the original microscope optical path beam 19 enters the coupling device through the top interface of the coupling device, and filters the stray light through the filter, and passes through the two-way The direction of the optical path does not change after the chromatic mirror, and the light beam of the optical tweezers capture module enters the coupling device through the light hole on the first support plate. At the same time, the light beam enters the microscope objective and is focused. At this time, a single optical trap will be formed at the focused position to achieve single cell capture.

The gravity-driven adjustable sample introduction device 3 includes a height-adjustable sample holder, a sample container, and a conduit, the conduit is connected to the sample container and the microfluidic chip 4, the sample container is mounted on the adjustable sample holder, and the adjustable sample The lifting and lowering of the rack drives the sample in the sample container to inject into the microfluidic chip 4 to realize the microflow of the sample solution;

The specific brightfield-optical tweezers single cell sorting method includes the following steps:

(1) Place the microfluidic chip on the microscope stage;

(2) inject the cell sample into the sample container, adjust the gravity drive to adjust the sample introduction device, and make the sample enter the microfluidic chip channel;

(3) Obtain the morphological image of the single cell through the bright field information of the microscope, process and analyze the morphological image through the computer, and identify the required single cell;

(4) Capture the single cell required by optical tweezers, and use the optical tweezers to move the single cell to the vicinity of the sampling point of the sample droplet;

(5) Push the single cell out of the channel through the sorting module to the droplet sampling point to form a single-cell droplet package;

(6) Use the capillary to wrap the droplets and export them into the capillary for later cultivation, expansion, and the like.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several improvements can be made without departing from the present invention, and these improvements should also be regarded as the present invention. The scope of protection of the utility model.

Claims (8)

1. A coupling device, characterized by: coupling device includes top interface, filter frame and dichroic mirror holder, the bottom interface, first backup pad and second backup pad, the top interface is placed with bottom interface parallel, connects through first backup pad and second backup pad, be equipped with logical unthreaded hole in the first backup pad, dichroic mirror holder installs in the second backup pad, the filter frame is installed on first backup pad, top interface, filter frame, dichroic mirror holder and bottom structure all are equipped with concentric round hole.

2. The coupling device of claim 1, wherein: the filter frame and the dichroic mirror frame are mounted through screws, and the mirror frame can be adjusted through manually or electrically rotating the screws.

3. The coupling device of claim 1, wherein: and a plurality of groups of filter mirror frames are arranged in the coupling device.

4. A micro-optical tweezers single cell sorting system, comprising:

-a microscope: for single cell signal detection;

-the coupling device of claim 1: the optical tweezers capture module is used for coupling the microscope and the optical tweezers;

-optical tweezers trapping module: for capturing and coordinating with a carrier platform to manipulate single cells;

-a sorting module: for single cell sorting;

-a computer: is respectively connected with the microscope and the optical tweezers capturing module and carries out program control.

5. The sorting system according to claim 4, wherein: the microscope is one of a bright field microscope, a fluorescence microscope or a Raman microscope.

6. The sorting system according to claim 4, wherein: the optical tweezers capturing module comprises single optical trap optical tweezers or multiple optical trap optical tweezers.

7. The sorting system according to claim 4, wherein: the microscope comprises an eyepiece imaging module, a microscope adapter plate, an objective microscope module and a microscope framework module, wherein the microscope adapter plate and the objective microscope module are arranged on a microscope framework; the top interface of the coupling device is connected with the eyepiece imaging module, the bottom interface of the coupling device is connected with the microscope adapter plate, and the optical tweezers capturing module is fixed on the microscope adapter plate.

8. The sorting system according to claim 4, wherein: the microscope comprises an eyepiece imaging module, a detection identification module, a microscope adapter plate, an objective microscope module and a microscope framework module, wherein the detection identification module is connected with the eyepiece imaging module, the microscope adapter plate and the objective microscope module are installed on a microscope framework, a top interface of the coupling device is connected with the detection identification module, a bottom interface of the coupling device is connected with the microscope adapter plate, and the optical tweezers capturing module is fixed on the microscope adapter plate.

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