CN212077049U - Coupling device and microscopic-optical tweezers single cell sorting system - Google Patents
Coupling device and microscopic-optical tweezers single cell sorting system Download PDFInfo
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- CN212077049U CN212077049U CN202020120685.4U CN202020120685U CN212077049U CN 212077049 U CN212077049 U CN 212077049U CN 202020120685 U CN202020120685 U CN 202020120685U CN 212077049 U CN212077049 U CN 212077049U
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Abstract
The utility model discloses a coupling device and it is micro-optical tweezers unicell sorting system, coupling device includes top interface, filter frame and dichroic mirror holder, the bottom interface, first backup pad and second backup pad, top interface and bottom interface parallel placement are connected through first backup pad and second backup pad, be equipped with logical unthreaded hole in the first backup pad, the dichroic mirror holder is installed 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. The optical tweezers can be simply and quickly introduced into the commercial microscope through the coupling device, and meanwhile, the sorting system is added, so that the microscope-optical tweezers sorting system and method are formed, the limitation that the commercial microscope is only used for observation is broken, and multiple functions are realized on one set of system: observation and observation of single cells, capture manipulation and separation extraction.
Description
Technical Field
The utility model relates to an optical instrument field, concretely relates to coupling device and it is micro-optical tweezers unicellular sorting system.
Background
Single cell analysis can reveal the diversity and difference of life basic unit-cell matter composition and physiological behavior, and is the mainstream leading-edge technology of life analysis at present. Single cell analysis is a study of differences among cell individuals in cell populations, including cell size, growth rate, chemical composition (phospholipids, proteins, metabolites, DNA/RNA), and the like, as well as the cause and mechanism of the generation of differences among cells. The research content relates to the fields of tumor biology, stem cells, microbiology, neurology, immunology and the like.
The biggest challenges in single cell analysis are represented by the tiny size of cells, and the problems of complex chemical composition, difficult composition trace and the like caused by small size. The size of a single cell is distributed in a micron scale or even a submicron scale, and extremely high requirements are provided for the manipulation of the single cell, the precision of an analysis instrument, the sensitivity and the like. Therefore, to realize single cell analysis, the following problems should be solved: single cell imaging, single cell signal acquisition, single cell capture, separation and extraction.
The optical microscope is an optical instrument which utilizes the optical principle to magnify and image tiny objects which cannot be distinguished by human eyes so as to extract fine structure information, and is a mark for human beings to enter the atomic era. The optical microscope is one of the common tools for single cell research and detection, and can observe the morphology and fluorescence characteristics of single cells to realize qualitative and quantitative research on the single cells.
However, the observation of single cells by an optical microscope is limited to observing cell morphology, cell structure, fluorescence specificity and the like, and the single cells cannot be manipulated, captured, separated and extracted, which greatly limits the subsequent operations (such as cell culture, single cell sequencing and the like) on the observed specific cells. And the optical microscope is a commercial product matured on the market, the optical path structure and the mechanical structure of the optical microscope are mature, and the introduction of a needed module on the basis of not influencing the performance of the microscope is one of difficulties.
SUMMERY OF THE UTILITY MODEL
Based on the prior art, the utility model aims at providing a coupling device makes and to introduce light tweezers unicellular sorting module on current microscope basis to the realization carries out the observation, the manipulation and the separation of unicellular simultaneously on an equipment.
The utility model provides a coupling device, coupling device includes top interface, filter frame and dichroic mirror holder, the bottom interface, first backup pad and second backup pad, top interface and bottom interface parallel placement are connected 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, filter frame installs on first backup pad, top interface, filter frame, dichroic mirror holder and bottom structure all are equipped with concentric round hole.
In another preferred embodiment, the filter holder and the dichroic mirror holder are mounted by screws, and the mirror holder is adjustable by manually or electrically rotating the screws.
In another preferred example, a plurality of groups of filter frames are arranged in the coupling device.
In another preferred embodiment, the top interface, the bottom interface, the first support plate and the second support plate are a unitary structure or a split structure.
In another preferred example, the light through hole on the first supporting plate is internally provided with screw threads.
In another preferred embodiment, the bottom interface and the bottom interface comprise a dovetail shape, a groove shape, or the like.
The utility model discloses another aspect provides a micro-optical tweezers unicellular sorting system, include:
-a microscope: for single cell signal detection;
-coupling means for: 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.
The microscope is one of a bright field microscope, a fluorescence microscope or a Raman microscope.
In another preferred example, the optical tweezers trapping module comprises a laser and a beam shaping device.
In another preferred example, the laser outputs laser light with one of 532nm, 785nm and 1064 nm.
In another preferred example, the wavelength of the laser output laser is 1064 nm.
In another preferred example, the beam shaping device is a beam expanding and collimating assembly.
In another preferred example, the laser is coaxial with the beam expanding and collimating assembly.
In another preferred example, the optical tweezers trapping module includes single optical trapping optical tweezers or multiple optical trapping optical tweezers.
In another preferred embodiment, the multi-optical trap optical tweezers are holographic optical tweezers.
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.
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.
In another preferred example, the detection and identification module is a fluorescence detection module or a raman detection module.
In another preferred embodiment, the sorting module comprises a microfluidic chip and a sample introduction device, and the microfluidic chip is placed on a microscope framework carrying platform.
In another preferred example, the sample injection device is a gravity-driven adjustment sample injection device, a syringe pump or a peristaltic pump.
In another preferred embodiment, the gravity-driven adjustment sampling device comprises a height-adjustable sample holder, a sample container and a conduit, wherein the conduit is connected with the sample container and the microfluidic chip, the sample container is mounted on the adjustable sample holder, and the lifting of the adjustable sample holder drives the sample in the sample container to be injected into the microfluidic chip, so that the micro-flow of the sample solution is realized.
The utility model has the advantages that:
(1) the limit of a mature optical path structure and a mechanical structure of a commercial microscope is broken through, and other modules are introduced into the microscope through a coupling device structure with a simple and compact structure on the basis of not influencing the performance of the microscope;
(2) by coupling the optical tweezers on a commercial microscope and adding the function of single cell sorting, the limitation that the microscope is only suitable for observation and monitoring is improved, and the capture operation, separation and extraction of the single cells are realized.
Drawings
In the accompanying drawings, like parts and features have like reference numerals. Many of the figures are schematic and may not be to scale.
FIG. 1 is a schematic diagram of a coupling device;
FIG. 2 is a schematic diagram of a micro-optical tweezers single cell sorting system;
FIG. 3 is a schematic diagram of an optical path of a single optical trap optical tweezers trapping module;
FIG. 4 is a schematic diagram of an optical path of a holographic optical tweezer trapping module;
FIG. 5 is a schematic diagram of a bright field-optical tweezers single cell sorting system.
The specific reference numbers are as follows:
1 a coupling device; 2, an optical tweezers capture module; 3, driving and adjusting a sample feeding device by gravity; 4, a micro-fluidic chip; 5 an image acquisition device; 6 ocular imaging module; 7, detecting and identifying the module; 8, a microscope adapter plate; 9 objective microscopic module; 10, a microscope framework; 11 a top interface; 12 a filter holder; 13 a first support plate; 14 a bottom port; 15 dichroic mirror mount; 16 a second support plate; 17 a laser; 18 a beam expanding collimation assembly; 19 light beam of original microscope light path; 20 microscope objective, 211/2 slide; 22 polarization beam splitter prism; 231/4 slide glass; 24 a spatial light modulator; 25 a first cemented doublet; 26 a mirror; 27 second doublet.
Detailed Description
To facilitate understanding of the embodiments of the present invention, the following description will be made in terms of several specific embodiments with reference to the accompanying drawings, and the embodiments are not intended to limit the embodiments of the present invention. Furthermore, the drawings are schematic and, thus, the present devices and apparatus are not limited by the size or scale of the schematic.
It is to be noted that in the claims and the description of the present patent, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, the use of the verb "comprise a" to define an element does not exclude the presence of another, same element in a process, method, article, or apparatus that comprises the element.
Example 1
Fig. 1 is a schematic structural diagram of a coupling device, which specifically includes:
Preferably, the filter holder 12 and the dichroic mirror holder 15 are mounted by screws, and the mirror holder is adjustable by adjusting the screws.
Preferably, a plurality of 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 a unitary structure or a separate structure.
Preferably, the light through hole of the first support plate 13 is internally provided with threads.
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 a microscope and an optical tweezers trapping module is shown in fig. 2:
the microscope comprises an eyepiece imaging module 6, a detection identification module 7, a microscope adapter plate 8, an objective microscope module 9 and a microscope framework module 10, wherein the detection identification module 7 is connected with the eyepiece imaging module 6, the microscope adapter plate 8 and the objective microscope module 9 are arranged on the microscope framework 10, a top interface of the coupling device 1 is connected with the detection identification module 7, a bottom interface is connected with the microscope adapter plate 8, and the optical tweezers capturing module 2 is fixed on the microscope adapter plate 8; the light beam of the original microscope light path enters the coupling device 1 through the top interface of the coupling device 1, the direction of the light path is not changed, the light beam of the optical tweezers capturing module 2 enters the coupling device 1 through the light through hole on the first supporting plate, the light beam is reflected to be overlapped with the light beam of the original microscope light path in space, the two light beams simultaneously enter the objective microscope module 9, and the focusing multiple of the objective microscope module 9 and a microscope carrying platform can be adjusted by a computer or manually to observe, capture and operate single cells.
Example 2
As shown in fig. 2, a fluorescence-optical tweezers single cell sorting system comprises:
-a fluorescence microscope, -a coupling device, -an optical tweezers trapping module, -a sorting module, -a computer (not shown in the figure);
the fluorescence microscope comprises an eyepiece imaging module 6, a detection identification module 7, a microscope adapter plate 8, an objective microscope module 9 and a microscope framework 10, wherein the eyepiece imaging module 6, the detection identification module 7, the objective microscope module 9 and the microscope framework 10 are commercial microscopes and can be freely disassembled and assembled, the detection identification module 7 is a fluorescence detection module, the detection identification module 7 is connected with the eyepiece imaging module 6, the microscope adapter plate 8 and the objective microscope module 9 are installed on the microscope framework 10, a top interface of the coupling device 1 is connected with the detection identification module 7, a bottom interface is connected with the microscope adapter plate 8, and the optical tweezers capturing module 2 is fixed on the microscope adapter plate; the sorting module comprises a microfluidic chip 4 and a gravity-driven adjusting sample introduction device 3, and the microfluidic chip 4 is placed on a microscope framework carrying platform.
The optical tweezers capturing module 2 is single optical trap optical tweezers, as shown in fig. 3: including a laser 17, a beam shaping device. The beam shaping device is a beam expanding and collimating component 18, and the optical path of the optical tweezers capturing module is shown in fig. 3: the light beam 19 of the original microscope light path enters the coupling device through the top interface of the coupling device, stray light is filtered through the optical filter, the direction of the light path is not changed after passing through the dichroic mirror, the light beam of the optical tweezers capturing module enters the coupling device through the light through hole in the first supporting plate, the light beam is reflected to be overlapped with the light beam of the original microscope light path in space after passing through the dichroic mirror, the two light beams simultaneously enter the microscope objective for focusing, and at the moment, a single light trap is formed at the focusing position, so that single cell capturing is realized.
In addition, the optical tweezers capturing module may also be multi-optical trap optical tweezers, including but not limited to holographic optical tweezers, and the specific structure is shown in fig. 4:
the laser 17 emits laser, the laser generates parallel light through the beam expanding collimation assembly 18, the parallel light passes through the 1/2 glass sheet 21 and the polarization splitting prism 22, the polarization direction of the incident light can be adjusted by adjusting the 1/2 wave plate 21 through the combination of the two, so that the incident light reaches the maximum transmittance after passing through the polarization splitting prism 22 and reaches the spatial light modulator 24 with the maximum light energy utilization rate, the spatial light modulator 24 is loaded with the computed hologram of the multiple light traps, the computed hologram is irradiated by the entered light and returns along the original light path, the computed hologram passes through the 1/4 glass sheet 23, the 1/4 glass sheet 23 is adjusted so that the light beam reaches the maximum contrast, the light beam which passes through the polarization splitting prism 22 again can reach the maximum reflectance, at this time, the computed hologram carrying information of the multiple light traps sequentially passes through the first cemented doublet 25, the reflecting mirror 26 and the second cemented doublet 27, the light beam 19 of the original microscope light path is superposed at the dichroic mirror of the coupling device and enters the microscope objective lens 20 for focusing, and at the moment, a plurality of light traps are formed at the focusing position to realize the capture of a plurality of single cells.
The gravity-driven adjusting sample introduction device 3 comprises a height-adjustable sample frame, a sample container and a conduit, wherein the conduit is connected with the sample container and the microfluidic chip 4, the sample container is arranged on the adjustable sample frame, the lifting of the adjustable sample frame drives the sample in the sample container to be injected into the microfluidic chip 4, and the micro-flow of a sample solution is realized.
Preferably, the sample injection device includes, but is not limited to, a gravity-driven adjustment sample injection device, a syringe pump, a peristaltic pump, or other sample injection devices.
The specific fluorescence-optical tweezers single cell sorting method comprises the following steps:
(1) placing the micro-fluidic chip on a microscope carrying platform;
(2) injecting a cell sample into a sample container, and adjusting gravity to drive and adjust a sample introduction device so that the sample enters a microfluidic chip channel;
(3) obtaining single cell fluorescence information through a fluorescence detection module, and processing a fluorescence image through a computer according to the single cell fluorescence information to identify a required sample;
(4) capturing the needed single cells through optical tweezers, and moving the single cells to the vicinity of a sample liquid drop sampling point by using the optical tweezers;
(5) pushing the single cell out of the channel to a droplet sampling point through a sorting module to form single cell droplet package;
(6) the liquid drops are wrapped and guided out by the capillary tube and enter the capillary tube for later culture, amplification and the like.
Example 3
As shown in fig. 3, a raman-optical tweezers single cell sorting system comprises:
-a raman microscope, -a coupling device, -an optical tweezers trapping module, -a sorting module, -a computer (not shown in the figure);
the Raman microscope comprises an eyepiece imaging module 6, a detection and identification module 7, a microscope adapter plate 8, an objective microscope module 9 and a microscope framework 10, wherein the eyepiece imaging module 6, the detection and identification module 7, the objective microscope module 9 and the microscope framework 10 are commercial microscopes and can be freely disassembled and assembled, the detection and identification module 7 is a Raman detection module, the detection and identification module 7 is connected with the eyepiece imaging module 6, the microscope adapter plate 8 and the objective microscope module 9 are arranged on the microscope framework 10, a top interface of the coupling device 1 is connected with the detection and identification module 7, a bottom interface is connected with the microscope adapter plate 8, and an optical tweezers capturing module 2 is fixed on the microscope adapter plate; the sorting module comprises a microfluidic chip 4 and a gravity-driven adjusting sample introduction device 3, and the microfluidic chip 4 is placed on a microscope framework carrying platform.
The optical tweezers capturing module 2 is single optical trap optical tweezers, as shown in fig. 3: including a laser 17, a beam shaping device. The beam shaping device is a beam expanding and collimating component 18, and the optical path of the optical tweezers capturing module is shown in fig. 3: the light beam 19 of the original microscope light path enters the coupling device through the top interface of the coupling device, stray light is filtered through the optical filter, the direction of the light path is not changed after passing through the dichroic mirror, the light beam of the optical tweezers capturing module enters the coupling device through the light through hole in the first supporting plate, the light beam is reflected to be overlapped with the light beam of the original microscope light path in space after passing through the dichroic mirror, the two light beams simultaneously enter the microscope objective for focusing, and at the time, a single light trap is formed at the focusing position to realize single cell capturing.
The gravity-driven adjustment sampling device 3 comprises a height-adjustable sample frame, a sample container and a guide pipe, wherein the guide pipe is connected with the sample container and the microfluidic chip 4, the sample container is arranged on the adjustable sample frame, and the adjustable sample frame is lifted to drive a sample in the sample container to be injected into the microfluidic chip 4, so that the micro-flow of a sample solution is realized;
the specific Raman-optical tweezers single cell sorting method comprises the following steps:
(1) placing the micro-fluidic chip on a microscope carrying platform;
(2) injecting a cell sample into a sample container, and adjusting gravity to drive and adjust a sample introduction device so that the sample enters a microfluidic chip channel;
(3) obtaining single cell Raman information through a Raman detection module, analyzing the obtained Raman spectrum signal through a computer or comparing the obtained Raman spectrum signal with a standard Raman spectrum signal in a constructed single cell phenotype database, and judging whether the single cell is the required single cell or not by giving an analysis result through the computer;
(4) capturing the needed single cells through optical tweezers, and moving the single cells to the vicinity of a sample liquid drop sampling point by using the optical tweezers;
(5) pushing the single cell out of the channel to a droplet sampling point through a sorting module to form single cell droplet package;
(6) the liquid drops are wrapped and guided out by the capillary tube and enter the capillary tube for later culture, amplification and the like.
Example 4
As shown in fig. 2, a bright field-optical tweezers single cell sorting system comprises:
-a bright field microscope, -a coupling device, -an optical tweezers trapping module, -a sorting module, -a computer (not shown in the figure);
the Raman microscope comprises an eyepiece imaging module 6, a detection and identification module 7, a microscope adapter plate 8, an objective microscope module 9 and a microscope framework 10, wherein the eyepiece imaging module 6, the objective microscope module 9 and the microscope framework 10 are commercial microscopes and can be freely disassembled and assembled, the microscope adapter plate 8 and the objective microscope module 9 are installed on the microscope framework 10, a top interface of the coupling device 1 is connected with the eyepiece imaging module 7, a bottom interface is connected with the microscope adapter plate 8, and the optical tweezers capturing module 2 is fixed on the microscope adapter plate; the sorting module comprises a microfluidic chip 4 and a gravity-driven adjusting sample introduction device 3, and the microfluidic chip 4 is placed on a microscope framework carrying platform.
The optical tweezers capturing module 2 is single optical trap optical tweezers, as shown in fig. 3: including a laser 17, a beam shaping device. The beam shaping device is a beam expanding and collimating component 18, and the optical path of the optical tweezers capturing module is shown in fig. 3: the light beam 19 of the original microscope light path enters the coupling device through the top interface of the coupling device, stray light is filtered through the optical filter, the direction of the light path is not changed after passing through the dichroic mirror, the light beam of the optical tweezers capturing module enters the coupling device through the light through hole in the first supporting plate, the light beam is reflected to be overlapped with the light beam of the original microscope light path in space after passing through the dichroic mirror, the two light beams simultaneously enter the microscope objective for focusing, and at the time, a single light trap is formed at the focusing position to realize single cell capturing.
The gravity-driven adjustment sampling device 3 comprises a height-adjustable sample frame, a sample container and a guide pipe, wherein the guide pipe is connected with the sample container and the microfluidic chip 4, the sample container is arranged on the adjustable sample frame, and the adjustable sample frame is lifted to drive a sample in the sample container to be injected into the microfluidic chip 4, so that the micro-flow of a sample solution is realized;
the specific bright field-optical tweezers single cell sorting method comprises the following steps:
(1) placing the micro-fluidic chip on a microscope carrying platform;
(2) injecting a cell sample into a sample container, and adjusting gravity to drive and adjust a sample introduction device so that the sample enters a microfluidic chip channel;
(3) obtaining a morphological image of the single cell through bright field information of a microscope, and processing and analyzing the morphological image through a computer to identify the required single cell;
(4) capturing the needed single cells through optical tweezers, and moving the single cells to the vicinity of a sample liquid drop sampling point by using the optical tweezers;
(5) pushing the single cell out of the channel to a droplet sampling point through a sorting module to form single cell droplet package;
(6) the liquid drops are wrapped and guided out by the capillary tube and enter the capillary tube for later culture, amplification and the like.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications can be made without departing from the scope of the present invention, and these modifications should also be regarded as the protection scope of the present invention.
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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