Imaging structure for cell sorting, cell sorting equipment and cell sorting method

Abstract

The invention discloses an imaging structure for cell sorting, cell sorting equipment and a cell sorting method, and belongs to the technical field of cell culture. The imaging structure comprises an illumination light source, the illumination light source is configured to be arranged at a preset height position, the illumination light source defines a channel for the pipette to pass through, and the pipette can penetrate through the channel to move in the direction close to or away from the container; the imaging module is arranged opposite to the illumination light source, and the imaging module is used for imaging a target position of the container from the lower part of the container; wherein an illumination light beam emitted by the illumination light source at least can illuminate a target position, and the pipette can move to the target position of the container to suck and / or spit out target cells according to an output result of the imaging module. According to the application, the position of the clone cell can be accurately identified, and the accuracy of clone cell selection is improved.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, and particularly to an imaging structure for cell sorting, a cell sorting device, and a cell sorting method. Background Art

[0002] Cell line development is the starting point and foundation of antibody drug CMC. During the cell line development process, since large-scale production must use highly homogeneous cloned cells in terms of genes and phenotypes, and at the same time prevent premature differentiation of stem cells during passage, and in addition, regulatory agencies only accept the production of biopharmaceuticals using cloned cells.

[0003] Currently, there are three commonly used methods for cell cloning and selection: The first is the limited dilution method, which means sucking out the cell line to be recloned from the culture well and performing cell counting to calculate the number of cells in 1 mL. It is often used to screen fused animal cells. The disadvantage of the limited dilution method is that it takes a long time, has low efficiency, high cost, high dependence on manual labor, and multiple rounds of dilution are required. The second is flow cytometry fluorescence sorting method. It can separate subpopulations of luminescent particles according to the fluorescence intensity and wavelength of the emitted light and can achieve monoclonal sorting. It can identify, classify, quantify, and separate cells in complex samples, and can simultaneously perform ultra-high-speed sorting and purification, high-throughput monoclonal sorting, or cell chip preparation on one to four specific cells at a time. The disadvantage of the flow cytometry fluorescence sorting method is that the cells are all dispersed, the tissue position information is lost, and it cannot be traced; it causes greater damage to cells and has a low cell survival rate. The third is the high-throughput screening method. High-throughput screening technology is a technical system based on experimental methods at the molecular and cell levels, using microplates as the experimental tool carrier, an automated operating system to execute the experimental process, a sensitive and fast detection instrument to collect experimental result data, a computer to analyze and process the experimental data, detecting tens of millions of samples at the same time, and supported by the corresponding database obtained. Its characteristic is that it has relatively high requirements for technology, the technology is monopolized by foreign countries, and the equipment price is high.

[0004] In related technologies, the patent with publication number CN111778161A discloses a method and device for selecting cloned cells. The method includes: obtaining a photo of a first multi-container containing cloned cells; selecting corresponding cloned cells from the photo according to the shape information of the cloned cells input by the user; generating a first control instruction for controlling the movement of the electric suction head component and the electric translation movement component in the cloned cell selection device according to the hole position of the selected cloned cells in the first multi-container, where the electric translation movement component carries the first multi-container; and controlling the suction head to suck the cloned cells in the hole position of the first multi-container according to the first control instruction.

[0005] In view of the related technologies mentioned above, the premise of high-precision equipment automation control is high accuracy in determining the position of cloned cells. The photos of the first multi-container containing cloned cells obtained by the above technologies are mainly taken by a microscopic photographing component. However, the cloned cells are located at the bottom of the container. When the microscopic photographing component takes pictures of the cloned cells from top to bottom, there is a lack of light source for the cloned cells located at the bottom of the container. At this time, the position of the cloned cells cannot be accurately identified, which in turn affects the subsequent precise selection of the cloned cells. Summary of the Invention

[0006] In order to help accurately identify the position of cloned cells and improve the accuracy of selecting cloned cells, the present application provides an imaging structure for cell sorting, a cell sorting device, and a cell sorting method.

[0007] The cell sorting device provided by the present application adopts the following technical solutions: First aspect An imaging structure for cell sorting, comprising: A lighting source, configured to be disposed at a preset height position, the lighting source defining a channel for a pipette to pass through, and the pipette can pass through the channel to move in a direction close to or away from the container; and An imaging module, disposed opposite to the lighting source, the imaging module being used to image a target position of the container from below the container; Wherein, the illumination beam emitted by the illumination source can at least illuminate the target position, and the pipette can move to the target position of the container to aspirate and / or eject target cells according to the output result of the imaging module.

[0008] By adopting the above technical solutions, the lighting source is disposed at a preset height position. This height position enables the illumination beam emitted by the lighting source to illuminate the target position without adjusting the position of the lighting source. At the same time, the position of the lighting source is fixed, and the position where the pipette moves relative to the lighting source can be more easily determined. Therefore, the height at which the pipette descends can be more precise, preventing the tip of the pipette from being damaged by the bottom of the container. In addition, the lighting source provides illumination transmitted light, which can completely illuminate the target cells, improve the imaging resolution of the target cells, shorten the imaging time, and the lighting design is biocompatible, which can reduce cell damage. Ultimately, the position of the target cells can be accurately identified, laying a foundation for the subsequent precise selection of cells.

[0009] Optionally, the lighting source is an annular light source, a semi-circular light source, a point light source, a line light source, a surface light source, or an arc light source.

[0010] Optionally, the lighting source is configured to be able to move along a preset path, and the preset path defines the channel; and / or When the pipette moves to the position of the target cell, the optical axis of the point light source passes through the tip position of the pipette.

[0011] By adopting the above technical solution, the point light source can move relative to the position of the pipette, so as to be able to specifically illuminate the position of the target cell, improve the illumination effect on the target cell, and further improve the accuracy of position recognition.

[0012] Optionally, the illumination light source includes a lamp board and a light-emitting chip arranged on the lamp board, and the light beam path of the light-emitting chip is inclined with respect to the central axis of the target position; and / or The illumination light source includes a lamp board and a light-emitting chip arranged on the lamp board, and the light-emitting chip is located in the channel through which the pipette passes.

[0013] By adopting the above technical solution, the brightness of the light source can be concentrated on the position of the target cell, so as to be able to accurately control the brightness of the light source of the target cell and further improve the accuracy of target cell position recognition.

[0014] Optionally, the illumination light source further includes a focusing lens arranged on the light-emitting path of the light-emitting chip. When the pipette moves to the position of the target cell, the focal point of the illumination light beam converges on the tip position of the pipette.

[0015] By adopting the above technical solution, the propagation path of the illumination light beam can be adjusted through the focusing lens, and further the brightness of the light source at the target position can be accurately adjusted according to requirements, improving the accuracy during the process of the pipette sucking and / or discharging the target cell.

[0016] Optionally, the preset height position is a fixed value, and the illumination range of the illumination light source is between 0.01 square millimeters and 20 square millimeters.

[0017] By adopting the above technical solution, when the imaging range is 0.1mm * 0.1mm, an illumination range of 0.01 square millimeters can perform precise imaging on the micro-pits.

[0018] Second aspect A cell sorting device, comprising: A carrier platform, the carrier platform is provided with an imaging window, a container is arranged on the carrier platform, and the carrier platform includes a vertical arm perpendicular to the carrier platform; and An imaging structure for cell sorting as described above, the imaging module is arranged under the carrier platform, and the imaging module can be aligned with the imaging window; the illumination light source is fixed to the vertical arm, and the pipette can move relative to the carrier platform.

[0019] By adopting the above technical solution, the container can be placed on the upper and lower sides of the imaging module and the illumination light source, and the pipette can move relative to the container, so as to aspirate or eject target cells. This solution reasonably sets the positions of various components, which is beneficial to the cell selection operation of the cell sorting device.

[0020] Optionally, a fixing member is fixed on the vertical arm, and an adjustment hole is formed in the fixing member. The adjustment hole is configured to adjust the position of the illumination light source in the XY plane; and / or the adjustment hole is configured such that the illumination light source can be detachably arranged on the fixing member.

[0021] By adopting the above technical solution, the position of the illumination light source can be easily adjusted through the fixing member, improving the convenience of adjusting the position of the light source.

[0022] Optionally, the cell sorting device further includes an XY-axis precision displacement stage arranged on the carrying platform and a positioning frame arranged on the XY-axis precision displacement stage. The container is arranged on the positioning frame, and the XY-axis precision displacement stage can carry the container to move to the target position.

[0023] By adopting the above technical solution, driving the container to move by using the XY-axis precision displacement stage can achieve the repeated positioning accuracy of the container, ensure the accuracy of the action, with high consistency, stability and reliability.

[0024] The third aspect A cell sorting method includes the following steps: Imaging: obtaining image information of the cell microchambers in the container through an imaging module; Intelligent recognition: identifying, analyzing and calculating the image information; Path planning: generating the movement trajectory of the pipette according to the result of intelligent recognition; Pipette movement: controlling the pipette to move to the target position according to the target movement trajectory; Scraping and aspiration: controlling the pipette to perform a scraping action to separate the adhered cells, and aspirating the separated cells by using the pipette; Post-aspiration imaging confirmation: imaging at the scraping position to confirm whether the cells are successfully aspirated; Transfer to a new well: transferring the target single cell to an empty cell chamber through the pipette; New-well imaging confirmation: imaging at the single-cell transfer position to confirm whether the cells are successfully transferred.

[0025] By adopting the above technical solution, each picking operation can be made reviewable and traceable, and individual cloned cells can be tracked throughout the process, assisting in optimizing the culture process, thereby achieving precise identification of cell passage, non-destructive precise picking and transfer of adherent cells, and greatly improving the selection efficiency.

[0026] In summary, this application includes at least one of the following beneficial technical effects: 1. The illumination light source is set at a preset height position. This height position enables the illumination light beam emitted by the illumination light source to illuminate the target position without the need to adjust the position of the illumination light source. At the same time, the position of the illumination light source is fixed, and the position where the pipette moves relative to the illumination light source can be more easily determined. Thus, the height at which the pipette descends can be more precise, preventing the tip of the pipette from being damaged by the bottom of the container. In addition, the illumination light source provides illumination transmitted light, which can completely illuminate the target cells, improve the imaging resolution of the target cells, shorten the imaging time, and the lighting design is biocompatible, which can reduce cell damage. Ultimately, the position of the target cells can be precisely identified, laying a foundation for the subsequent precise picking of cells.

[0027] 2. The point light source can move relative to the position of the pipette, so as to be able to specifically illuminate the position of the target cells, improve the illumination effect on the target cells, and further improve the accuracy of position recognition.

[0028] 3. The propagation path of the illumination light beam can be adjusted through the focusing lens, and thus the light source brightness at the target position can be precisely adjusted according to requirements, improving the precision during the process of the pipette sucking and / or discharging the target cells.

[0029] 4. Each picking operation can be made reviewable and traceable, and individual cloned cells can be tracked throughout the process, assisting in optimizing the culture process, thereby achieving precise identification of cell passage, non-destructive precise picking and transfer of adherent cells, and greatly improving the selection efficiency.

[0030] 5. This application locks the focal plane of the imaging module lens to the plane of the pipette needle and can automatically track and focus according to the up and down movement of the pipette.

[0031] 6. This application uses a customized lateral light source to avoid the shadow of the pipette needle and avoid imaging interference on the upper part of the pipette needle, and records the picking process in real time.

[0032] 7. It can realize real-time monitoring of the whole process of single-cell picking, realize the picking tracking and confirmation of single cells, and can realize the morphological tracking of each link in the picking process, including the state capture in the pipette pipeline.

[0033] 8. The device may include a cell culture environment system. A sealed space is set in the device to provide an environment suitable for cell growth with constant temperature, humidity, and gas ratio, so as to realize in-situ selection and culture of cells. Description of the Drawings

[0034] Figure 1 It is a schematic structural diagram of the imaging structure of the present application.

[0035] Figure 2 It is a schematic structural diagram of the imaging structure of the present application where the illumination light source is a point light source.

[0036] Figure 3 It is a schematic structural diagram of the imaging structure of the present application where the light beam path of the light-emitting chip is inclined with respect to the central axis of the target position.

[0037] Figure 4 It is a schematic structural diagram of the imaging structure of the present application where the light-emitting chip is located in the channel through which the pipette passes.

[0038] Figure 5 It is a schematic structural diagram of the imaging structure of the present application where a focusing lens is further provided on the light output path of the light-emitting chip.

[0039] Figure 6 It is a schematic overall structural diagram of the cell sorting device of the present application.

[0040] Figure 7 is Figure 6 an enlarged schematic diagram of part A in

[0041] Description of the Reference Numerals: 10, container; 11, pipette; 20, illumination light source; 201, point light source; 202, lamp board; 203, light-emitting chip; 21, channel; 22, focusing lens; 30, imaging module; 40, carrier table; 41, imaging window; 42, XY-axis precision displacement stage; 43, positioning bracket; 50, vertical arm; 51, fixing member; 52, adjustment hole; 60, lead screw moving mechanism; 61, clamping plate. Detailed Description of the Embodiments

[0042] The following further describes the present application in detail with reference to the attached Figures 1-7 drawings.

[0043] In the related art, the container 10 is a well plate for culturing or collecting target cells. The well plate can be a 12-well plate or a 96-well plate, and the number of wells in the well plate is not limited. A culture groove is formed in the well plate. The culture groove can be a round groove or a polygonal groove, etc. A nano-well plate is placed in the culture groove, and the nano-holes of the nano-well plate can accommodate cell suspension.

[0044] The pipette 11 is used to aspirate or eject target cells. The pipette 11 is connected to a micro-aspiration system, and the aspiration accuracy of the cell suspension is ±5 nl. The micro-aspiration system is not the focus of this application and will not be elaborated here.

[0045] An embodiment of the present application discloses an imaging structure for cell sorting. Refer to Figure 1 , the imaging structure for cell sorting includes an illumination light source 20 and an imaging module 30. The illumination light source 20 and the imaging module 30 are respectively located on the upper and lower sides of the container 10. The illumination light source 20 provides transmitted light from top to bottom for the target position of the container 10, and the imaging module 30 images the target position of the container 10 from bottom to top. The imaging module 30 can be a microscopic imaging module, which has the characteristics of high resolution and efficient imaging, and can reduce the number of photos and shorten the imaging time while ensuring high resolution.

[0046] A channel 21 for the pipette 11 to pass through is defined in the center of the illumination light source 20, and the pipette 11 can pass through the channel 21 to move in a direction closer to or away from the container 10. The movement direction of the pipette 11 can be vertical up and down movement or inclined up and down movement.

[0047] The illumination light source 20 is set at a preset height position, and the preset height position is a fixed value. The preset height value is the distance between the nano-well plate and the illumination light source 20, and this distance can be 30 mm. At this height position, the illuminated field of view of the illumination light source 20 ranges from 0.01 square millimeters to 20 square millimeters. At this time, the illumination beam emitted by the illumination light source 20 can illuminate the target position of the container 10, so that the pipette 11 can move to the target position of the container 10 to aspirate or eject target cells according to the output result of the imaging module 30.

[0048] The illumination light source 20 provides illumination transmitted light, which can completely illuminate the target cells, improve the imaging resolution of the target cells, shorten the imaging time, and the illumination design has biocompatibility, which can reduce cell damage, and finally can accurately identify the position of the target cells.

[0049] Refer to Figure 1 and Figure 2 , wherein, the illumination light source 20 can be an annular light source, a semi-circular light source, a point light source 201, a line light source, a surface light source or an arc light source.

[0050] In a preferred embodiment, the illumination light source 20 is a point light source 201 which can move circumferentially along the channel 21, so as to be able to adjust the position of the point light source 201 in real time, enable the optical axis of the point light source 201 to pass through the tip position of the pipette 11, and thus be able to specifically illuminate the position of the target cell, improving the illumination effect on the target cell. The position adjustment of the point light source 201 can be driven and adjusted by a conventional mechanical structure, and the conventional mechanical structure will not be elaborated here.

[0051] Referring to Figure 3 , in another preferred embodiment, the illumination light source 20 includes a lamp board 202 and light-emitting chips 203 arranged on the lamp board 202. The illumination light source 20 can be an annular light source or a semi-circular light source. At this time, a plurality of light-emitting chips 203 are arranged, and the light beam paths of the plurality of light-emitting chips 203 are inclined with respect to the central axis of the target position. Such an arrangement can concentrate the light source brightness on the position of the target cell, and thus can precisely control the light source brightness of the target cell.

[0052] Referring to Figure 4 , in another preferred embodiment, the illumination light source 20 includes a lamp board 202 and light-emitting chips 203 arranged on the lamp board 202. The light-emitting chips 203 are located in the channel 21 through which the pipette 11 passes. This solution blocks the light beam through the channel 21, so that the light beam irradiates to the target position along the direction of the channel 21, reducing the propagation loss of the light beam.

[0053] Referring to Figure 5 , in another preferred embodiment, a focusing lens 22 is further arranged on the light-emitting path of the light-emitting chip 203. The focusing lens 22 can converge the focus of the illumination light beam at the tip position of the pipette 11. The focusing lens 22 can be a plano-convex focusing lens 22, a biconvex focusing lens 22, an aspherical focusing lens 22, etc. The focusing lens 22 can also be a fixed-type focusing lens 22, that is, the focusing lens 22 has been pre-adjusted in position and angle. At this time, the focus of the illumination light beam converges at the preset position. The focusing lens 22 can also be a non-fixed-type focusing lens 22. An angle adjustment mechanism is used to adjust the angle of the focusing lens 22, and the focusing lens 22 is used to adjust the position of the focus of the illumination light beam. By means of the focusing lens 22, the propagation path of the illumination light beam can be adjusted, and further the light source brightness at the target position can be accurately adjusted according to requirements, improving the accuracy during the process of the pipette 11 sucking and / or discharging the target cell.

[0054] The implementation principle of an imaging structure for cell sorting in an embodiment of the present application is as follows: The illumination light source 20 is set at a preset height position, which enables the illumination light beam emitted by the illumination light source 20 to illuminate the target position without adjusting the position of the illumination light source 20, simplifying the position adjustment steps of the illumination light source 20. At this time, the illumination light source 20 provides illumination transmitted light, which can completely illuminate the target cells, improve the imaging resolution of the target cells, shorten the imaging time, and the illumination design has biocompatibility, which can reduce cell damage. Eventually, the position of the target cells can be accurately identified, laying a foundation for the precise selection of subsequent cells.

[0055] An embodiment of the present application also discloses a cell sorting device. Refer to Figure 6 , the cell sorting device includes a carrier table 40, an imaging window 41 is opened on the carrier table 40, an imaging module 30 is located at the imaging window 41, an XY-axis precision displacement stage 42 is provided on the carrier table 40, and a positioning frame 43 is provided on the XY-axis precision displacement stage 42. The container 10 is arranged on the positioning frame 43, and the XY-axis precision displacement stage 42 can move the container 10 to the target position.

[0056] When the container 10 moves to the target position, the imaging module 30 is aligned with the imaging window 41, and the imaging light beam emitted by the imaging module 30 passes through the imaging window 41 and irradiates the bottom of the container 10. The bottom of the container 10 is a transparent plate. At the same time, the illumination light beam emitted by the illumination light source 20 irradiates into the container 10 from top to bottom, and precisely micro-images the cells in the container 10.

[0057] Refer to Figure 6 and Figure 7 , wherein, a vertical arm 50 perpendicular to the tabletop of the carrier table 40 is fixed on the side of the carrier table 40, a fixing member 51 is fixed on the vertical arm 50, and the fixing member 51 can be a fixing plate, and an adjustment hole 52 is opened on the fixing plate. In this embodiment, the adjustment hole 52 is two intersecting long strip holes, and the photo light source is fixed on the fixing plate through the adjustment hole 52, and the position of the illumination light source 20 in the XY plane can be adjusted through the adjustment hole 52; and the illumination light source 20 can be detachably arranged on the fixing plate through the adjustment hole 52.

[0058] In other embodiments, the adjustment hole 52 can also be multiple intersecting long strip holes, such as four vertically and horizontally intersecting long strip holes, etc.

[0059] At the same time, a lead screw moving mechanism 60 for driving the pipette 11 to move up and down is also provided on the vertical arm 50. A clamping plate 61 is installed at the output end of the lead screw moving mechanism 60. The pipette 11 is clamped by the clamping plate 61, and the pipette 11 can move up and down through the channel 21 of the illumination light source 20. The lead screw moving mechanism 60 is used to drive the pipette 11 to lift along the Z axis, so that the pipette 11 can move out of or into the container 10 to aspirate or eject the target.

[0060] In another embodiment, an A-axis rotation mechanism may further be provided between the clamping plate 61 and the pipette 11. The A-axis rotation mechanism is used to drive the pipette 11 to rotate along the A-axis, and the A-axis is a vertical line perpendicular to the tabletop of the carrier 40. At this time, the tip direction of the pipette 11 is aligned with the scraping direction, and the pipette 11 performs precise rotation along the A-axis, so that precise scraping operation can be achieved.

[0061] The implementation principle of a cell sorting device according to an embodiment of the present application is as follows: The cell sorting device of the present application can realize accurate identification of cell passage, non-destructive and precise selection and transfer of adherent cells, can monitor and obtain data on key variables during the culture and passage processes, provides valuable data for process optimization, and can be used in multiple biomedical fields such as stem cell passage and differentiation selection, tumor tissue cell culture, organoid formation and orthogonal culture, and colony orthogonal culture.

[0062] An embodiment of the present application also discloses a cell sorting method. The method includes the following steps: S1. Dilution: The dilution can be performed by an artificial dilution method.

[0063] Specifically, 1. Manually add the culture medium into the culture tank of the well plate. The well plate can be a 12-well plate or a 96-well plate, and the number of wells of the well plate is not limited; 2. Then place the nano-microwell plate into the culture tank; 3. Manually use a pipette to aspirate the cell suspension in the centrifuge tube; 4. Add the cell suspension to the nano-microwell plate in a dropwise manner, and add it drop by drop into the micro-wells of the nano-microwell plate in an array manner, so that the surface of the nano-microwell plate is all dripped with the cell suspension.

[0064] S2. Imaging: Obtain the image information of the cell microchambers in the nano-microwell plate through an imaging system. The image information includes the morphology of each cell microchamber in the nano-microwell plate. The morphology of the cell microchambers includes single-cell chambers, multi-cell chambers, and empty cell chambers.

[0065] Specifically, the imaging step includes: 1. Taking the center of the nano-microwell plate as the coordinate origin, the XY-axis precision displacement stage 42 for placing the well plate is controlled by a program to move. Each time it moves one step (in the X direction or the Y direction), and the step is a multiple of the pitch of the nano-holes. The center of the nano-holes for each imaging coincides with the field of view center of the imaging module 30.

[0066] 2. During the movement of the well plate, the imaging module 30 located at the bottom of the well plate takes pictures of the cell microchambers. During the picture-taking process, the camera and lens of the imaging module 30 will automatically lift and lower for focusing to make the imaging of cells at different heights clearer.

[0067] 3. Each time the orifice plate moves relative to the imaging module 30, the imaging module 30 performs a photographing action until the imaging module 30 acquires images of all the holes of the nano-microwell plate.

[0068] 4. Perform algorithmic stitching and processing on the images of all the holes of the nano-microwell plate to form a complete image information, and the image information includes the morphology of each cell microchamber of the nano-microwell plate. The morphology of the cell microchamber includes single-cell chambers, multi-cell chambers, and empty cell chambers.

[0069] S3. Intelligent recognition: Recognize, analyze, and calculate the image information.

[0070] Specifically, after the image is synthesized and meets the requirements of intelligent recognition, the algorithm performs overall recognition, analysis, and calculation on the image, including: 1. Automatically calculate the confluence rate. The confluence rate is the percentage of the area occupied by cells and the area of the bottom of the well plate that can be occupied by cells, and count the number of cells; 2. Analyze the health status through cell morphology and color, and output the recognized block diagram. This block diagram precisely classifies the cells to distinguish the area cells most suitable for passage; 3. Calculate the number of single-cell chambers, and determine whether the number of single-cell chambers meets the expectation. If it meets, there is no need to perform the scraping and aspiration step; if it does not meet, the scraping and aspiration step needs to be performed.

[0071] S4. Path planning: After intelligent recognition, the program starts the trajectory planning algorithm to generate the movement trajectory of the pipette 11 needle tip. The purpose of planning the trajectory is to enable the pipette 11 to accurately reach the position of the target cells.

[0072] S5. Move the multi-cell chamber to a preset position: Control the XY-axis precision displacement stage 42 to move, so that the multi-cell chamber of the nano-microwell plate moves into the field of view of the imaging module 30.

[0073] S6. Pipette and spread evenly: The pipette 11 moves to the multi-cell chamber, and the pipette 11 is used to pipette and spread the multi-cells to make the cells spread evenly, which is convenient for better aspiration of single cells.

[0074] S7. Scraping and aspiration: Control the pipette 11 to perform a scraping action to separate the adhered cells, and use the pipette 11 to aspirate the separated cells.

[0075] Specifically, align the tip direction of the pipette 11 tube head with the scraping direction, and rotate around the vertical line as the rotation axis to achieve the scraping action, so as to achieve precise operation.

[0076] S8. Imaging confirmation after aspiration: After the pipette 11 has completed scraping and aspiration, the imaging module 30 images the scraping position to confirm whether the cells have been successfully aspirated. This operation is used to confirm that the transferred cells are correct and do not exceed the transfer range, ensuring that each operation is verifiable and traceable.

[0077] S9. Transfer to a new well: Transfer single cells to an empty cell chamber through the pipette 11 to form a single-cell chamber.

[0078] Specifically, the program controls the precise displacement stage 42 of the XY axis to move, aligning the pipette 11 with the empty cell chamber, and controlling the pipette 11 to eject one cell into the empty cell chamber to form a single-cell chamber.

[0079] S10. Imaging confirmation of the new well: Image the single-cell transfer position to confirm whether the cells have been successfully transferred.

[0080] Specifically, the imaging module 30 images the transfer position to confirm whether the cells have been successfully transferred and do not exceed the transfer range, ensuring that each operation is verifiable and traceable.

[0081] Through the cell sorting method of the present application, each operation can be made verifiable and traceable, the whole process of individual cloned cells can be tracked, the culture process can be assisted in optimization, so as to achieve accurate identification of cell passage, non-destructive precise selection and transfer of adherent cells, greatly improving the selection efficiency.

[0082] The above are all preferred embodiments of the present application. The protection scope of the present application is not limited thereby. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application should be covered within the protection scope of the present application.

Claims

1. An imaging structure for cell sorting, characterized in that, Comprising: A lighting light source (20), configured to be set at a preset height position, the lighting light source (20) defining a channel (21) for a pipette (11) to pass through, and the pipette (11) can pass through the channel (21) to move in a direction close to or away from a container (10); And An imaging module (30), disposed opposite to the lighting light source (20), and the imaging module (30) is used to image a target position of the container (10) from below the container (10); Wherein, the lighting beam emitted by the lighting light source (20) can at least illuminate the target position, and the pipette (11) can move to the target position of the container (10) to aspirate and / or eject target cells according to the output result of the imaging module (30).

2. The imaging structure for cell sorting according to claim 1, wherein: The lighting light source (20) is an annular light source, a semi-circular light source, a point light source (201), a line light source, a surface light source or an arc light source.

3. The imaging structure for cell sorting according to claim 2, characterized in that: The lighting light source (20) is configured to be movable along a preset path, and the preset path defines the channel (21); and / or When the pipette (11) moves to the position of the target cell, the optical axis of the point light source (201) passes through the tip position of the pipette (11).

4. An imaging structure for cell sorting according to any one of claims 1 to 3, characterized in that: The lighting light source (20) includes a lamp board (202) and a light-emitting chip (203) disposed on the lamp board (202), and the light beam path of the light-emitting chip (203) is inclined with respect to the central axis of the target position; and / or The lighting light source (20) includes a lamp board (202) and a light-emitting chip (203) disposed on the lamp board (202), and the light-emitting chip (203) is located in the channel (21) through which the pipette (11) passes.

5. An imaging structure for cell sorting according to claim 4, characterized in that: The lighting light source (20) further includes a focusing mirror (22) disposed on the light-emitting path of the light-emitting chip (203). When the pipette (11) moves to the position of the target cell, the focus of the lighting beam converges at the tip position of the pipette (11).

6. The imaging structure for cell sorting according to claim 1, characterized in that: The preset height position is a fixed value, and the illuminated field of view of the lighting light source (20) ranges from 0.01 square millimeters to 20 square millimeters.

7. A cell sorting device, characterized in that, Comprising: A carrier (40), the carrier (40) is provided with an imaging window (41), a container (10) is disposed on the carrier (40), and the carrier (40) includes an upright arm (50) perpendicularly disposed to the carrier (40); and An imaging structure for cell sorting according to any one of claims 1-6, wherein the imaging module (30) is disposed below the carrier (40) and the imaging module (30) can be aligned with the imaging window (41); the lighting light source (20) is fixed to the upright arm (50), and the pipette (11) can move relative to the carrier (40).

8. The cell sorting device according to claim 7, characterized in that: A fixing member (51) is fixed on the vertical arm (50), and an adjustment hole (52) is formed in the fixing member (51). The adjustment hole (52) is configured to adjust the position of the illumination light source (20) in the XY plane; and / or the adjustment hole (52) is configured such that the illumination light source (20) can be detachably arranged on the fixing member (51).

9. A cell sorting device according to claim 7, characterized in that: The cell sorting device further includes an XY-axis precision displacement stage (42) arranged on the carrying platform (40) and a positioning frame (43) arranged on the XY-axis precision displacement stage (42). The container (10) is arranged on the positioning frame (43), and the XY-axis precision displacement stage (42) can carry the container (10) to move to a target position.

10. A cell sorting method based on the cell sorting device according to any one of claims 7-9, characterized in that, It includes the following steps: Imaging: Obtaining image information of the cell microchambers in the container (10) through the imaging module (30); Intelligent recognition: Identifying, analyzing, and calculating the image information; Path planning: Generating a movement trajectory of the pipette (11) according to the result of the intelligent recognition; Movement of the pipette (11): Controlling the pipette (11) to move to the target position according to the target movement trajectory; Scraping and sucking: Controlling the pipette (11) to perform a scraping action to separate the adhered cells, and sucking the separated cells by the pipette (11); Imaging confirmation after sucking: Imaging at the scraping position to confirm whether the cells are successfully sucked; Transferring to a new hole: Transferring the target single cell to an empty cell chamber through the pipette (11); Imaging confirmation of the new hole: Imaging at the single cell transfer position to confirm whether the cells are successfully transferred.

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