CN114350474B - Automatic sorting device and sorting method for single cells - Google Patents

Abstract

The application discloses a single cell sorting device, which comprises an imaging unit, an optical tweezers coupling module, a microscopic imaging objective lens, an injection pump and a two-dimensional motion platform; the collecting device comprises a receiver, a one-dimensional sliding table and a supporting frame; the one-dimensional sliding table drives the receiver to move up and down; the support frame is provided with a chip clamp for mounting the microfluidic chip, and the receiver is provided with a container for accommodating single cells sorted by the microfluidic chip. A method for sorting single cells, comprising the steps of: preparation stage, B: sorting stage, C: and (3) ending stage. The single cells can be rapidly sorted, and the working efficiency is improved.

Description

Automatic sorting device and sorting method for single cells

Technical Field

The present application relates to the field of single cell sorting, and in particular, to an automatic sorting apparatus and a sorting method for single cells.

Background

Single cell analysis can reveal the basic unit of life-cell material composition, diversity and variability of physiological behaviors, and is the mainstream leading-edge technology of life analysis today. Single cell analysis is a study in cell populations, which is directed to cell-to-cell individual differences including cell size, growth rate, chemical composition (phospholipids, proteins, metabolites, DNA/RNA), etc., as well as the cause and mechanism of cell-to-cell differences. The research content relates to the fields of tumor biology, stem cells, microbiology, nervous system science, immunology and the like.

At present, a micro-droplet wrapped single cell sorter (EasySort) is adopted for single cell sorting, which is a combination of micro-optical tweezers and a microfluidic droplet wrapping technology, is a lossless single cell sorting method, and is beneficial to downstream experiments, namely single cell culture and MDA.

Aiming at the above technology: the existing EasySort has the problems that the sorting process needs more manual operation, the automation degree is low, and the working efficiency is affected.

Disclosure of Invention

In a first aspect, the present application provides a single cell sorting apparatus, which adopts the following technical scheme:

a single cell sorting device comprises an imaging unit, an optical tweezers coupling module, a microscopic imaging objective lens, an injection pump and a two-dimensional motion platform; the method is characterized in that: the collecting device comprises a receiver and a supporting frame;

the one-dimensional sliding table drives the receiver to move up and down;

the support frame is provided with a chip clamp for mounting the microfluidic chip, and the receiver is provided with a container for accommodating single cells sorted by the microfluidic chip.

Through adopting above-mentioned technical scheme, the up-and-down motion of one-dimensional slip table drive receiver can make the better single cell that the micro-fluidic chip of container divides accept, and through the reciprocates of one-dimensional slip table control container, need not the manual work to remove, can make the operation more accurate simple, improves work efficiency.

Optionally, one-dimensional slip table includes the supporting seat of vertical setting and fixed connection at the cylinder of supporting seat upper surface, and the cylinder can drive the receiver up-and-down motion.

Through adopting above-mentioned technical scheme, the main driving method of one-dimensional slip table is the cylinder, through the up-and-down motion of gas gang6 drive receiver, can make the more stable of receiver motion, avoids influencing the collection of single cell.

Optionally, the two ends of the microfluidic chip are respectively and fixedly connected with a hose and a capillary, the hose is connected with an injection pump, the capillary is vertically arranged, and the axes of the capillary and the container can be overlapped.

Through adopting above-mentioned technical scheme, the hose makes things convenient for micro-fluidic chip and syringe pump to connect, makes the syringe pump provide fluid for micro-fluidic chip, and the capillary can lead the liquid that contains single cell, and the capillary can stretch into in the container to can avoid the liquid drop that contains cell to fall the in-process and float in the outside of container.

Optionally, a sliding groove is formed in one side wall of the supporting seat, a sliding block is arranged in the sliding groove, and the tail end of the telescopic rod of the air cylinder extends into the sliding groove and is fixedly connected with the sliding block;

a moving platform is arranged below the receiver and is fixedly connected with the sliding block, and the cylinder drives the moving platform to move up and down to indirectly drive the receiver to move up and down;

the upper surface of the mobile platform is provided with a concave accommodating groove, a driving motor is fixedly connected in the accommodating groove, and an output shaft of the driving motor is fixedly connected with the receiver.

Through adopting above-mentioned technical scheme, driving motor on the mobile platform can drive the receiver and rotate, and then the container on the convenient drive receiver leaves or is close to the capillary, conveniently takes off or installs the container, only needs to install the container, does not need the manual work to go to align container and capillary, also does not need the manual work to go to lift the receiver and is the container, can improve work efficiency.

Optionally, the receiver is disc-shaped or rectangular, and a plurality of blind holes are formed in the receiver and uniformly distributed around the central point of the receiver;

the number of the containers is multiple, and the containers are sequentially installed in the blind holes.

Through adopting above-mentioned technical scheme, a plurality of blind holes have been seted up on the receiver, all have the container in every blind hole, so install a plurality of containers on the receiver, the container can rotate simultaneously, so only need artifical batch change container can, the receiver is under driving motor and one-dimensional slip table drive, can rise automatically and descend and rotate, and then can make the container align with the capillary in proper order automatically, the capillary can be to a cell of input in every container, and then can accomplish many times cell sorting automatically, work efficiency has been improved by a wide margin.

In a second aspect, the present application provides a single cell sorting method, which adopts the following technical scheme:

a method for sorting single cells comprises the following steps: the method comprises the following steps:

a: the preparation stage, a microfluidic chip and a container for accommodating single cells are selected, and the microfluidic chip, the capillary and the container are processed;

b: a sorting stage, namely starting to sort the samples and sorting out single cells;

c: and in the ending stage, stopping and sorting the used instrument, taking the container away, and collecting and summarizing the sorted single cells.

Through adopting above-mentioned technical scheme, through above-mentioned step, can be quick accurate select separately the cell, ensure to select out single cell, and collect the induction to single cell, take single cell in the convenience follow-up experiment and carry out experiment or analysis.

Optionally, the preparation phase includes the steps of:

(1) selecting a microfluidic chip, vacuumizing the microfluidic chip, preparing a sample, and determining a container for accommodating single cells as a PCR tube;

(2) selecting an adopted instrument;

(3) the PCR tubes are multiple, oil phase or liquid phase is filled in the PCR tubes, and the multiple PCR tubes are sequentially placed in blind holes of the receiver;

(4) the microfluidic chip is arranged on the chip clamp, and then the chip clamp is arranged on the supporting frame;

(5) connecting all structures of the instrument;

(6) firstly, a syringe pump is used for flushing a channel on a microfluidic chip, and the time T=10 min;

(7) cleaning the outer wall of the capillary tube for 3-5 minutes;

(8) the imaging unit is adjusted to make the image displayed by the imaging unit clear, and sorting is started.

Through adopting above-mentioned technical scheme, can carry out accurate processing and preparation to sample, the instrument that the experiment adopted through step (1) - (8), prepare before the separation, wash the passageway on capillary and the micro-fluidic chip, can avoid the debris that adheres on capillary or the micro-fluidic chip to cause the influence to the effect of cell separation.

Optionally, in the step (3), when the liquid in the PCR tube is an oil phase, specifically a mineral oil, and when the liquid in the PCR tube is a liquid phase, specifically an aqueous solution or a cell liquid.

Through adopting above-mentioned technical scheme, can select the liquid type of packing into in the PCR pipe according to actual conditions, can be better hold the single cell that selects out, can also avoid the solution in the PCR pipe to cause the influence to single cell follow-up experiment or analysis.

Optionally, in the step (5), when the structures of the apparatus are connected, the flow rate of the syringe pump is set to V.

By adopting the technical scheme, the injection pump is set with proper flow rate, so that the fluid is ensured to have flow force, single cells can be taken away, the impact of the excessive flow rate on the cells can be avoided, and the damage of the cells is avoided.

Optionally, the sorting stage may use a manual mode for sorting, where the manual mode sorting includes the following steps:

(1) amplifying a sample through a microscope objective lens, uploading an image into an imaging interface, and manually identifying target single cells to be sorted;

(2) manually operating a two-dimensional motion platform to enable optical tweezers points in the optical tweezers coupling module to move on target cells, starting the optical tweezers, and capturing the target cells;

(3) the path is manually planned to avoid the rest cells, and the target cells are moved to the position M, so that the fluid provided by the injection pump is convenient for carrying the target cells in an impact manner;

(4) starting a collecting device to collect the single cells conveyed by the capillary tube;

(5) repeating steps (1) - (4) until all containers on the receptacle contain a single cell cutoff.

By adopting the technical scheme, the cells can be better sorted through the steps, and the sorted cells can be ensured to be intact by adopting manual control.

Alternatively, the sorting stage may employ a semi-automatic mode of sorting, the semi-automatic mode of sorting comprising the steps of:

(1) the imaging unit processes, identifies and uploads the image transmitted by the microscope, so that an operator can observe the image conveniently;

(2) manually sorting through an image unit to sort out target single cells;

(3) automatically dragging, namely automatically dragging a single cell to an M position;

(4) the collection device can automatically collect liquid drops containing single cells.

Through adopting above-mentioned technical scheme, through above-mentioned step, can be quick select separately the cell, the manual work is selected separately the cell, confirms suitable single cell of selecting separately, ensures that the cell is not impaired cell, then pulls automatically, collects automatically, can improve work efficiency, still can not select separately impaired cell, avoids influencing the experiment or the analysis of follow-up single cell.

Optionally, the sorting stage may adopt a fully automatic mode for sorting, and the fully automatic mode sorting includes the following steps:

(1) full-automatic identification;

(2) full-automatic dragging;

(3) and (5) full-automatic collection.

Through adopting above-mentioned technical scheme, can carry out automatic identification to single cell through above-mentioned step and select separately, drag, collect, and then realize the purpose of automatic separation single cell, can increase substantially work efficiency.

In view of the above-mentioned, it is desirable,

1. the receiver can be driven to rotate and move up and down through the one-dimensional sliding table, the moving platform and the driving motor arranged on the moving platform, and a plurality of containers can be arranged on the receiver, so that when single separated cells are collected, the sequential automatic collection of the containers can be realized, the containers are only needed to be replaced manually in batches, the work of collecting the cells by a single operation container is not needed, and the working efficiency can be further improved;

2. through the step A: preparation stage, B: sorting stage, C: in the ending stage, single cells can be better collected, and the single cells can be accurately collected and generalized, so that the subsequent experiment or analysis by using the single cells is convenient.

Drawings

FIG. 1 is a schematic diagram showing the overall structure of a cell sorting apparatus according to the first embodiment.

Fig. 2 is a schematic overall structure of the collecting assembly in the first embodiment.

Fig. 3 is an exploded view showing the relationship between the chip holder and the supporting frame in the first embodiment.

Fig. 4 is a schematic diagram showing a chip holder structure in the first embodiment.

Fig. 5 is a schematic view highlighting the connection block in the first embodiment.

Fig. 6 is a schematic diagram of the highlighting driving motor in an embodiment of the first embodiment.

Fig. 7 is an outflow block diagram of the sorting method in the second embodiment.

Fig. 8 is a schematic diagram of the principle of sorting microfluidic chips in the second embodiment.

Reference numerals illustrate:

1. an imaging unit; 11. a fluorescent module; 12. an optical tweezers coupling module; 13. an adapter plate; 14. a microscope skeleton; 15. a microimaging objective; 16. a two-dimensional motion platform; 17. a syringe pump; 2. a collection module; 2. a collection module; 3. a bottom plate; 4. a collection assembly; 41. one-dimensional sliding table; 411. a support base; 412. a cylinder; 413. a chute; 414. a slide block; 42. a mobile platform; 421. a receiving groove; 422. a driving motor; 43. a receiver; 431. a blind hole; 5. a support frame; 51. a riser; 52. a support plate; 521. a step; 53. a fixing plate; 54. a fixing bolt; 6. a chip clamp; 61. a rectangular plate; 611. a positioning groove; 612. a through hole; 613. positioning holes; 62. a compacting sheet; 621. an adjusting bolt; 622. a spring; 63. a relief groove; 64. a connecting block; 641. a left inverted cone opening; 642. a right inverted cone opening; 7. a microfluidic chip; 71. a hose; 72. a capillary tube; 8. a cell; 81. optical tweezers points; 82. and (5) a PCR tube.

Detailed Description

Embodiment one;

referring to fig. 1, most of the current cell sorting apparatuses include an imaging unit 1, a fluorescent module 11, an optical tweezers coupling module 12, an adapter plate 13, a microscope skeleton 14, a microscopic imaging objective 15, a two-dimensional motion platform 16, a syringe pump 17, and a collection module 2. The imaging unit 1, the fluorescent module 11, the adapter plate 13, the microscope skeleton 14, the microscopic imaging objective 15 and the two-dimensional motion platform 16 jointly form a microscopic imaging unit, the microscopic imaging unit is used for acquiring single cell image signals, and the two-dimensional platform 16 is used for driving the microscopic imaging objective 15 to move back and forth and left and right on a plane, so that the imaging unit is more convenient for capturing images; the single cell image signal includes: morphology size, fluorescent signal, etc.; the optical tweezers coupling module 12 is used for capturing single cells; the syringe pump 17 provides a fluid with fluidity, which is used to drive the movement of the single cells, and the collection module 2 is used to collect the single cells.

Referring to fig. 2, the automatic collection module 2 includes a base plate 3, a collection assembly 4 provided on the base plate 3, a support frame 5, and a chip fixture 6 provided on the support frame 5. The bottom plate 3 is used for installing on the cell sorter to make the installation of whole automatic collection module, support frame 5 is used for supporting chip anchor clamps 6, has the micro-fluidic chip 7 to press from both sides on the chip anchor clamps 6, collects the liquid that contains single cell that the subassembly 4 was carried to micro-fluidic chip 7.

Referring to fig. 3, the support frame 5 includes two risers 51 fixed on the bottom plate 3 and a support plate 52 fixedly connected to the risers 51, the support plate 52 is transversely fixedly connected to the upper end of the risers 51, and the chip clamp 6 is installed between the two support plates 52, and the support frame 5 can raise and support the chip clamp 6, so that the microfluidic chip 7 and the collection assembly 4 mounted on the chip clamp 6 are conveniently matched.

Referring to fig. 3, the upper surface of the side wall, which is close to the two support plates 52, is provided with a step 521, and two sides of the chip clamp 6 are located on the step 521, so that the two support plates 52 can support and limit the chip clamp 6, and the probability of movement of the chip clamp 6 is reduced.

The one end that backup pad 52 is close to receiver 43 is equipped with fixed plate 53, and the length direction of fixed plate 53 and the length direction mutually perpendicular setting of backup pad 52, and fixed plate 53 can laminate the upper surface at chip anchor clamps 6 one end, and the both ends of fixed plate 53 are lapped respectively at the upper surface of two backup pads 52, and peg graft at the both ends of fixed plate 53 has fixing bolt 54, and fixing bolt 54's end and backup pad 52 threaded connection. Under the action of the fixing bolts 54, the fixing plates 53 are fastened to the two support plates 52, and thereby the chip clamp 6 is fixed in an auxiliary manner.

Referring to fig. 3, a hose 71 and a capillary 72 are connected to the microfluidic chip 7, the hose 71 provides fluid to the microfluidic chip 7, and the fluid entering the microfluidic chip 7 can carry away the single cells sorted by the microfluidic chip 7, and the capillary 72 guides the fluid so that the fluid containing the single cells enters the collection assembly 4.

Referring to fig. 3 and 4, the chip holder 6 includes a rectangular plate 61 and a pressing piece 62 provided on the rectangular plate 61; rectangular positioning grooves 611 are formed in the rectangular plate 61, rectangular through holes 612 are formed in the bottom surface of the positioning grooves 611, the through holes 612 are smaller than the positioning grooves 611, the positioning grooves 611 play a role in positioning when the microfluidic chip 7 is mounted, the rectangular through holes 612 are used for the flexible tubes 71 to pass through, positioning holes 613 are formed in the bottom surface of the positioning grooves 611, the positioning holes 613 can be used for the capillary tubes 72 to pass through, and meanwhile the positioning holes 613 can also be used for supporting and positioning the capillary tubes 72.

Referring to fig. 4, the upper surfaces of the rectangular plates 61 at two sides of the positioning groove 611 are provided with symmetrical yielding grooves 63, the yielding grooves 63 are communicated with the positioning groove 611, and two side walls of the microfluidic chip 7 are conveniently clamped through the yielding grooves 63, so that clamping and placing of the microfluidic chip 7 are completed.

Referring to fig. 4, the compressing plate 62 is above the positioning groove 611, the length direction of the compressing plate 62 is perpendicular to the length direction of the positioning groove 611, one end of the compressing plate 62 is on the upper surface of the rectangular plate 61, an adjusting bolt 621 is inserted into the end of the compressing plate 62, the adjusting bolt 621 passes through the compressing plate 62 to be in threaded connection with the rectangular plate 61, a spring 622 is sleeved on the adjusting bolt 621, and the upper end and the lower end of the spring 622 are respectively abutted against the upper surface of the compressing plate 62 and the lower surface of the nut of the adjusting bolt 621. The adjusting bolt 621 is rotated to enable the spring 622 to bear or not bear any more, whether the compressing sheet 62 applies pressure to the micro-fluidic chip 7 is controlled, the micro-fluidic chip 7 is conveniently installed or taken down, the adjusting bolt 621 applies pressure to the compressing sheet 62 through the spring 622, the spring 622 can play a role in buffering, and the condition that the compressing sheet 622 is damaged due to overlarge pressure caused by overlarge screwing of the adjusting bolt 621 can be avoided.

Referring to fig. 4 and 5, the lower surface of the rectangular plate 61 is also fixedly connected with a connection block 64, two ends of the connection block 64 are respectively fixedly connected with a left back taper port 641 and a right back taper port 642, the right back taper port 642 is used for inserting and connecting the hose 71, and the left back taper port 641 is used for connecting with the syringe pump 17, so that the syringe pump 17 supplies fluid to the microfluidic chip 7.

Referring to fig. 3 and 6, the collecting assembly 4 includes a one-dimensional sliding table 41, a moving platform 42, and a receiver 43, the one-dimensional sliding table 41 is mounted on the base plate 3, the one-dimensional sliding table 41 drives the moving platform 42 to move up and down, the receiver 43 is mounted on the moving platform 42, and a tubular container can be placed on the receiver 43, where the tubular container is used for receiving the fluid conveyed by the capillary tube 72.

Referring to fig. 3 and 6, the one-dimensional sliding table 41 comprises a supporting seat 411 and an air cylinder 412 fixedly connected to the upper end of the supporting seat 411, the air cylinder 412 is vertically arranged downwards, a sliding groove 413 is formed in the side wall, close to the moving platform 42, of the supporting seat 411, a sliding block 414 is arranged in the sliding groove 413, the sliding block 414 is fixedly connected with the moving platform 42, and a piston rod of the air cylinder 412 extends into the sliding groove 413 to be fixedly connected with the sliding block 414; so that the one-dimensional sliding table 41 can drive the moving platform 42 to move up and down, and further the container on the receiver 43 can conveniently receive the fluid conveyed by the capillary 72.

Referring to fig. 3 and 6, a recessed receiving groove 421 is formed in the upper surface of the moving platform 42, a driving motor 422 is fixedly connected in the receiving groove 421, and an output shaft of the driving motor 422 is fixedly connected with the receiver 43, so that the receiver 43 can be rotated under the driving of the driving motor 422. The receiver 43 is in a disc shape, a plurality of blind holes 431 are formed in the edge of the upper surface of the receiver 43, the blind holes 431 are uniformly distributed around the axis of the receiver 43, a tubular container is placed in the blind holes 431, and can rotate along with the receiver 43, so that after the microfluidic chip 7 finishes sorting single cells, fluid drives the cells to flow through the capillary 72 and enter the container, when one container is received, the driving motor 422 drives the receiver 43 to rotate, so that a new container is aligned with the capillary 72, the fluid containing the single cells is received after the capillary 72 is conveyed again, continuous automatic fluid receiving is realized, the container does not need to be replaced all the time manually, and the working efficiency is improved.

The receiver 43 in this example may be rectangular, and the blind holes 431 only need to be uniformly distributed annularly around the center point of the receiver 43, and the output shaft of the driving motor 422 is fixedly connected with the center of the receiver 43.

The one-dimensional sliding table 41 in this embodiment may also be a two-dimensional sliding table, and the two-dimensional sliding table drives the receiver 43 to reciprocate on a horizontal plane, so that the receiver 43 may also be a twenty-four hole plate, or may be a rectangular plate, two rows of blind holes 431 or array distributed blind holes 431 are formed on the upper surface of the receiver 43, and only the blind holes 431 on the receiver 43 need to be moved to the capillary 72, so that a container installed in the blind holes 431 may be aligned with the capillary 72.

The specific use mode of the embodiment is as follows: firstly, mounting a microfluidic chip 7 on a chip clamp 6, then inserting a hose 71 into a left back taper port 641, fixing and stabilizing, and then connecting the hose 71 with a syringe pump 17 through a right back taper port 642, wherein the syringe pump 17 provides fluid for the microfluidic chip 7; the capillary 72 on the microfluidic chip 7 is aligned through the positioning hole 613 and the receptacle on the receptacle 43, and the axis of the capillary 72 coincides with the axis of the blind hole 431, thereby enabling better alignment of the capillary 72 with the receptacle.

The micro-fluidic chip 7 can sort the cells 8, so that grabbing of the single cells 8 is realized, and fluid drives the single cells 8 to flow, so that sorting of the single cells 8 is realized; the fluid containing single cells 8 flows into the capillary 72, at this time, the one-dimensional sliding table 41 is started, the air cylinder 412 controls the moving platform 42 to ascend, and then drives the receiver 43 to ascend, so that the container on the receiver 43 ascends, the lower end of the capillary 72 stretches into the container, the fluid flowing out of the capillary 72 can more accurately fall into the container, and the fluid flowing out of the capillary 72 is tiny and is easily influenced by factors such as airflow flowing because the diameter of the capillary 72 is small and the single cells 8 are invisible to naked eyes, so that the fluid cannot enter the container, and the lower end of the capillary 72 stretches into the container, so that the fluid can be prevented from falling outside the container.

When the fluid flowing out of the capillary 72 enters the container, the separation of the single cells 8 is completed, at this time, the air cylinder 412 in the one-dimensional sliding table 41 controls the receiver 43 to descend, the container is separated from the capillary 72, then the driving motor 422 is started to drive the receiver 43 to rotate, so that a new container is aligned with the capillary 72, the one-dimensional sliding table 41 drives the receiver 43 to ascend again, the lower end of the capillary 72 enters the new container, the container is used for receiving and collecting the single cells 8, the above steps are repeated, the collection of the single cells 8 can be automatically completed for a plurality of times, only the containers on the receiver 43 need to be replaced manually, the containers do not need to be held manually to be received in sequence, the collision between the containers and the capillary 72 can be avoided while the working efficiency is improved, and the damage probability of the capillary 72 is reduced.

Embodiment two;

a method of sorting individual cells 8, referring to fig. 7, comprising the steps of:

a: a preparation stage, wherein the adopted equipment is processed before sorting, and a sorted sample is prepared;

referring to fig. 7, the preparation phase includes the steps of:

(1) selecting a microfluidic chip 7, vacuumizing the microfluidic chip 7, preparing a sample, and determining a container for accommodating single cells 8 as a PCR tube 82;

(2) the instrument used is selected, and comprises an imaging unit 1, a fluorescence module 11, an optical tweezers coupling module 12, an adapter plate 13, a microscope imaging framework, a two-position motion platform, a collecting assembly 4, a microscope imaging objective lens and a syringe pump 17;

(3) the number of the PCR tubes 82 is multiple, the PCR tubes 82 are filled with liquid, and the PCR tubes 82 are sequentially placed on the receiver 43;

when the liquid in the PCR tube 82 is an oil phase, the liquid is specifically mineral oil;

when the liquid in the PCR tube 82 is in a liquid phase, the liquid is specifically an aqueous solution or a cell 8 liquid;

(4) the microfluidic chip 7 is mounted on the chip holder 6, and then the chip holder 6 is mounted on the support frame 5;

(5) connecting all structures of the instrument, and setting the flow speed V of the injection pump 17 to be 1uL/min-2uL/min;

(6) firstly, the injection pump 17 washes the channel on the micro-fluidic chip 7 for a period of time t=1 min;

(7) cleaning the outer wall of the capillary 72 for 3-5 minutes;

(8) the imaging unit 1 is adjusted so that the image displayed by the imaging unit 1 is clear, and sorting is started.

When the microfluidic chip 7 and the capillary 72 are cleaned, two PCR tubes 82 are taken as waste liquid tubes to receive and rinse waste liquid, so that the waste liquid is prevented from polluting the receiver 43.

B: a sorting stage, namely starting to sort the samples, and sorting single cells 8;

referring to fig. 7 and 8, the sorting stage uses a manual mode for sorting, the manual mode comprising the steps of:

(1) amplifying a sample through a microscope objective lens, uploading an image into an imaging interface, and manually identifying target single cells 8 to be sorted;

(2) manually operating the two-dimensional motion platform 16 to enable the optical tweezers points 81 in the optical tweezers coupling module 12 to move on the target cells 8, opening the optical tweezers, and capturing the target cells 8;

(3) the rest cells 8 are avoided by manually planning paths, and the target cells 8 are moved to the position M, so that the fluid provided by the injection pump 17 is convenient for carrying the target cells 8 in an impact manner;

(4) starting the collection assembly 4 to collect the single cells 8 conveyed by the capillary 72;

(5) steps (1) - (4) are repeated until all containers on receptacle 43 hold one single cell 8.

C: in the ending stage, stopping the instrument and finishing; the PCR tube 82 on the receiver 43 is removed and the syringe pump 17 is stopped; the PCR tube 82 is collected, generalized and stored, so that the single cells 8 in the PCR tube 82 can be conveniently used for subsequent experiments or analysis.

The movement process of the collecting assembly 4 in the step B is that the one-dimensional sliding table 41 moves up and down, and the liquid drops wrapped with the single cells 8 at the tail ends of the capillaries 72 are dropped into a container on the receiver 43, namely a PCR tube 82, and the oil phase liquid is filled in the PCR tube 82 to form water-in-oil liquid drops wrapped with the single cells 8; the one-dimensional slide table 41 performs the up-and-down movement, and the receiver 43 rotates to move the new PCR tube 82 again to the position right below the capillary 72.

In this embodiment, it is also disclosed that the sorting stage in step B may employ a semiautomatic mode, including the following steps:

(1) the imaging unit 1 processes, identifies and uploads the image transmitted by the microscope, so that an operator can observe the image conveniently;

(2) manually sorting through an image unit to sort out target single cells 8;

(3) automatic dragging, namely automatically dragging the single cell 8 to an M position;

(4) the collection assembly 4 is capable of automatically collecting droplets containing individual cells 8.

In the step (3), the micro-fluidic chip 7 is driven to move in the X, Y direction by the movement of the two-dimensional motion platform 16 in the X, Y direction, and the positions of the optical tweezers in the optical tweezers coupling module 12 are unchanged, so that the automatic dragging of the single cells 8 is realized.

The embodiment also discloses that the sorting stage in the step B can adopt a full-automatic mode, and the specific working process is as follows:

the imaging unit can automatically identify a target single cell, and move an optical tweezer point in the optical tweezer coupling module 12 to a target cell position through the two-dimensional motion platform 16, at the moment, the optical tweezer coupling module 12 is started to capture the target cell, the target cell is automatically dragged to a point M in fig. 8 through a pre-planned path, at the moment, the collecting assembly is started, and the collecting assembly collects the target cell; the actions of the collection assembly are: the one-dimensional sliding table moves up and down for a specified distance, the moving completion disc rotates through one blind hole position, and the circulation is sequentially carried out until all blind hole positions are collected.

The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes in structure, shape and principle of this application should be covered in the protection scope of this application.

Claims (8)

1. The single cell sorting device comprises an imaging unit (1), an optical tweezers coupling module (12), a microscopic imaging objective lens (15), an injection pump (17) and a two-dimensional motion platform (16); the method is characterized in that: the device also comprises a collecting assembly (4), wherein the collecting assembly (4) comprises a receiver (43) and a supporting frame (5);

the one-dimensional sliding table (41) drives the receiver (43) to move up and down;

a chip clamp (6) is arranged on the support frame (5), the chip clamp (6) is used for installing the microfluidic chip (7), a container is arranged on the receiver (43), and the container is used for accommodating single cells (8) separated by the microfluidic chip (7);

the one-dimensional sliding table (41) comprises a supporting seat (411) which is vertically arranged and an air cylinder (412) which is fixedly connected to the upper surface of the supporting seat (411), and the air cylinder (412) can drive the receiver (43) to move up and down;

two ends of the microfluidic chip (7) are fixedly connected with a hose (71) and a capillary tube (72), the hose (71) is connected with an injection pump (17), the capillary tube (72) is vertically arranged, and the axes of the capillary tube (72) and the container can be overlapped;

a sliding groove (413) is formed in one side wall of the supporting seat (411), a sliding block (414) is arranged in the sliding groove (413), and the tail end of a telescopic rod of the air cylinder (412) extends into the sliding groove (413) and is fixedly connected with the sliding block (414);

a movable platform (42) is arranged below the receiver (43), the movable platform (42) is fixedly connected with a sliding block (414), and an air cylinder (412) drives the movable platform (42) to move up and down and indirectly drives the receiver (43) to move up and down;

the upper surface of the moving platform (42) is provided with a concave accommodating groove (421), a driving motor (422) is fixedly connected in the accommodating groove (421), and an output shaft of the driving motor (422) is fixedly connected with the receiver (43).

2. The single-cell sorting apparatus according to claim 1, wherein: the receiver (43) is disc-shaped or rectangular, a plurality of blind holes (431) are formed in the receiver (43), and the blind holes (431) are uniformly distributed around the center point of the receiver (43);

the number of containers is plural, and the containers are sequentially mounted in the blind holes (431).

3. A method of sorting individual cells based on the sorting apparatus of any one of claims 1-2, characterized in that: the method comprises the following steps:

a: a preparation stage, namely selecting a microfluidic chip (7) and a container for accommodating single cells (8), and processing the microfluidic chip (7), the capillary (72) and the container;

b: a sorting stage, in which sorting of the sample is started to sort out single cells (8);

c: and in the ending stage, the used instrument is stopped and tidied, the container is taken away, and the sorted single cells (8) are collected and summarized.

4. A method of sorting individual cells according to claim 3, wherein: the preparation phase comprises the following steps:

(1) selecting a microfluidic chip (7), vacuumizing the microfluidic chip (7), preparing a sample, and determining a container for accommodating single cells (8) as a PCR tube (82);

(2) selecting an adopted instrument;

(3) the number of the PCR tubes (82) is multiple, oil phase or liquid phase is filled in the PCR tubes (82), and the PCR tubes (82) are sequentially placed in the blind holes (431) of the receiver (43);

(4) the microfluidic chip (7) is arranged on the chip clamp (6), and then the chip clamp (6) is arranged on the supporting frame (5);

(5) connecting all structures of the instrument;

(6) firstly, a syringe pump (17) washes a channel on a micro-fluidic chip (7), and the time T=1 min;

(7) cleaning the outer wall of the capillary tube (72) for 3-5 minutes;

(8) the imaging unit (1) is adjusted to make the image displayed by the imaging unit (1) clear, and sorting is started.

5. The method for sorting single cells according to claim 4, wherein: in the step (3), when the liquid in the PCR tube (82) is an oil phase, specifically a mineral oil, and when the liquid in the PCR tube (82) is a liquid phase, specifically an aqueous solution or a cell (8) liquid.

6. The method for sorting single cells according to claim 5, wherein: in the step (5), when the structures of the instrument are connected, the flow rate of the injection pump (17) is set to be V.

7. A method of sorting individual cells according to claim 3, wherein: the sorting stage can adopt a manual mode for sorting, and the manual mode sorting comprises the following steps:

(1) amplifying the sample by a microscope objective and uploading the image into an imaging interface, and manually identifying target single cells (8) to be sorted;

(2) manually operating a two-dimensional motion platform (16) to enable an optical tweezer point (81) in an optical tweezer coupling module (12) to move to a target cell (8), opening optical tweezers and capturing the target cell (8);

(3) the path is manually planned to avoid the rest cells (8), and the target cells (8) are moved to the position M, so that the fluid provided by the injection pump (17) is convenient to impact and carry the target cells (8);

(4) starting a collection assembly (4) to collect single cells (8) conveyed by the capillary (72);

(5) repeating steps (1) - (4) until all containers on the receptacle (43) hold one single cell (8) closed.

8. A method of sorting individual cells according to claim 3, wherein: the sorting stage can adopt a semi-automatic mode for sorting, and the semi-automatic mode sorting comprises the following steps:

(1) the imaging unit (1) processes, identifies and uploads the image transmitted by the microscope, so that an operator can observe the image conveniently;

(2) manually sorting out target single cells (8) by an image unit;

(3) automatically dragging, namely automatically dragging the single cells (8) to an M position;

(4) the collection assembly (4) is capable of automatically collecting droplets containing individual cells (8).