Automated scanning, picking device and method for rare cell single cell manipulation

Abstract

The application discloses an automatic scanning and picking device and method for single-cell operation of rare cells, and comprises the following: an automatic multi-color fluorescence scanning and recognizing module, the automatic multi-color fluorescence scanning and recognizing module comprising a multi-channel fluorescence microscope, a planar sliding table being installed on the multi-channel fluorescence microscope, and the multi-channel fluorescence microscope being connected with an image acquisition and analysis system; a multi-angle console, the multi-angle console comprising a base, the base being installed on the planar sliding table, a rotating assembly, a first pitching assembly and a second pitching assembly being installed on the base, a micro-operation platform, the micro-operation platform comprising a Z-axis sliding table installed on the multi-channel fluorescence microscope, and a capillary glass microneedle being fixed at the tail end of an execution end of the Z-axis sliding table; a nanometer microcavity chip, the nanometer microcavity chip being placed on an object table; and an upper computer, the upper computer being used for controlling the operation of the whole device. The application greatly improves the position stability in the process of processing of rare cells, and is suitable for the characteristics of low quantity and difficult operation of the rare cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection and single-cell analysis technology, and in particular to an automated scanning and picking device and method for single-cell manipulation of rare cells. Background Technology

[0002] Single-cell level genomic and transcriptomic analysis has become an important tool for tumor heterogeneity research and precision medicine. Circulating tumor cells (CTCs), as rare cells in peripheral blood, can reflect the polyclonal evolution characteristics of primary and metastatic lesions, and are of great value for dynamic monitoring and drug resistance mechanism research. To achieve single-cell sequencing and functional studies of CTCs, it is first necessary to reliably classify cells into single-cell states under a microscope and to be able to quickly and accurately locate and pick target cells. Currently, existing single-cell manipulation platforms each have their shortcomings, and microplates combined with flow cytometry sorting or microscopic pick-up methods are preferred. The current method suffers from several drawbacks. Due to the large volume of the micropores, the cells settle randomly, making it difficult to achieve stable and regular single-cell localization, and the degree of automation is limited. Although traditional planar microfluidic chips can achieve continuous flow screening, shear forces can easily cause cells to detach or migrate during immunostaining within the chip. At the same time, the three-dimensional position of cells within the flow channel is not fixed, increasing the difficulty of automatic identification and accurate pickup. Commercial single-cell dispensing systems mostly focus on the random distribution or encapsulation of suspended cells, and lack specially optimized structural designs for adherent / settled cells that have already undergone immunostaining, especially the very few CTCs.

[0003] To address the aforementioned technical issues, this invention provides an automated scanning and picking device and method for single-cell manipulation of rare cells. Summary of the Invention

[0004] The purpose of this invention is to provide an automated scanning and picking device and method for single-cell manipulation of rare cells, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: The present invention provides an automated scanning and picking device for single-cell manipulation of rare cells, comprising: An automatic multicolor fluorescence scanning and recognition module, comprising a multi-channel fluorescence microscope, wherein a planar sliding stage is mounted on the multi-channel fluorescence microscope, and the multi-channel fluorescence microscope is connected to an image acquisition and analysis system; A multi-angle control console includes a base, which is mounted on a planar slide. A rotation component, a first pitch component, and a second pitch component are mounted on the base. The first pitch component is mounted on the rotation component, and the second pitch component is mounted on the first pitch component. A platform is mounted on the second pitch component. The micro-manipulation platform includes a Z-axis slide mounted on the multi-channel fluorescence microscope. A capillary glass microneedle is fixed to the end of the execution end of the Z-axis slide, and the rear end of the capillary glass microneedle is connected to an adjustable pressure microinjector. A nano-microcavity chip, wherein the nano-microcavity chip is placed on the stage; The host computer is used to control the operation of the overall device.

[0006] The automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention includes, in which the rotating component comprises: A rotating column, which is vertically rotatably connected to the top of the base; The first gear set is rotatably connected to the platform, and the rotating column is fixed on the first gear set; A first adjustment knob is rotatably connected to the platform and fixed to one of the gears on the first gear set.

[0007] According to the automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention, the first pitch component includes a mounting frame, the mounting frame being rotatably connected to the top of the rotating column via a mounting shaft, a second gear set being mounted on the rotating column via a fixing frame, the second gear set being fixed to the mounting shaft, and a second adjusting knob being rotatably connected to the fixing frame, the second adjusting knob being fixed to one set of gears in the second gear set.

[0008] According to the automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention, the second pitch assembly includes a pitch arm rotatably connected to the mounting frame, the stage is mounted on the pitch arm, a third gear set is mounted on the mounting frame, the third gear set is axially connected to the pitch arm, and a third adjustment knob is mounted on the mounting frame, the third adjustment knob is fixed to one set of gears of the third gear set.

[0009] According to the automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention, the nanocavity chip includes a base layer and a top microchannel layer. The base layer is detachably mounted on the planar slide. A plurality of microcavities arranged in a two-dimensional array are processed on the top surface of the base layer. An intermediate isolation layer is provided on the top surface of the base layer. A flow channel is arranged on the top microchannel layer. An inlet and an outlet are provided on the top microchannel layer. The inlet and the outlet are respectively connected to the flow channel, and the flow channel is connected to the microcavity.

[0010] The automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention has an inner diameter of 5–25 μm for the capillary glass microneedles.

[0011] In the automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention, the step resolution of the planar slide in the XY direction is preferably no greater than 1 μm.

[0012] According to the automatic scanning and picking device for single-cell manipulation of rare cells provided by the present invention, the lateral dimension of the microcavity on the nanocavity chip is 10–40 μm and the depth is 10–30 μm.

[0013] An automated scanning and picking method for single-cell manipulation of rare cells includes the following steps: Step 1: Turn on the host computer and start the entire device. Perform system self-checks on the automatic multicolor fluorescence scanning and recognition module, multi-angle control console, and micro-operation platform to ensure that each module is operating normally and the signal connection is stable. Drop the sample containing rare cells into the designated area of ​​the nanocavity chip, ensuring that the sample is evenly spread and does not overflow the chip area. Then, place the nanocavity chip stably on the stage of the multi-angle control console and adjust the chip position to be in the center of the stage area for easy subsequent scanning and operation. At the same time, set the basic parameters of the multi-channel fluorescence microscope through the host computer, adjust the adjustable pressure microinjector, remove air bubbles from the capillary glass microneedle to ensure that the microneedle is unobstructed, preset the appropriate suction pressure and release pressure, and complete the device initialization and sample preparation. Step two: The host computer controls the rotation component, first pitch component, and second pitch component of the multi-angle control console to adjust the nanocavity chip on the stage at multiple angles, ensuring that the chip surface is vertically aligned with the objective lens of the multi-channel fluorescence microscope to ensure the clarity of the fluorescence scan. Then, the host computer controls the planar slide to move the multi-channel fluorescence microscope. Based on the size of the nanocavity chip, the scanning area is set, and the scanning start and end points are clearly defined to avoid missed or redundant scans. At the same time, the focal length of the multi-channel fluorescence microscope is adjusted to clearly image the cell samples on the chip, preparing for subsequent fluorescence scanning and cell identification. Step 3: Activate the automatic multicolor fluorescence scanning and recognition module. The host computer controls the multi-channel fluorescence microscope to perform omnidirectional automatic multicolor fluorescence scanning of the sample on the nano-microcavity chip according to the preset scanning path and parameters. The planar slide moves the microscope at a uniform speed to ensure that there are no blind spots or repetitions in the scanning process. During the scanning process, the image acquisition and analysis system acquires cell images of each fluorescence channel in real time, preprocesses the acquired images to remove background noise and optimize image contrast, and then extracts cell contours through image recognition algorithms, calculates the average fluorescence intensity value of each fluorescence channel, and automatically identifies the target rare cells by combining the fluorescence characteristic parameters of rare cells. At the same time, it records the specific coordinate position of each rare cell on the nano-microcavity chip and feeds back the recognition results and coordinate information to the host computer to complete the automatic identification and positioning of rare cells. Step four: Based on the identified rare cell coordinates, the host computer synchronously controls the planar slide and multi-angle control console to move the target rare cell directly below the capillary glass microneedle on the micromanipulation platform. Simultaneously, the stage angle is finely adjusted via the rotation component, the first pitch component, and the second pitch component to ensure the rare cell is in the optimal picking position for the microneedle. Then, the Z-axis slide of the micromanipulation platform is moved downwards, causing the capillary glass microneedle to slowly approach the target rare cell. When the microneedle tip reaches a preset distance, the host computer controls the adjustable pressure microinjector to activate the negative pressure suction function, gently adsorbing the target rare cell through the capillary glass microneedle. After adsorption, the Z-axis slide is moved upwards, causing the microneedle and cell to rise together, completing the picking operation for a single rare cell. The suction pressure is monitored in real time during the picking process to prevent excessive pressure from damaging cell viability. Step 5: According to experimental requirements, the host computer controls the coordinated movement of the planar slide, multi-angle control console, and Z-axis slide to move the capillary glass microneedle containing rare cells to the pre-set cell collection container. Adjust the microneedle position to ensure the tip is aligned with the designated area of ​​the collection container. Then, the host computer controls the adjustable pressure microinjector to switch to positive pressure release mode, and the rare cells in the capillary glass microneedle are smoothly released into the collection container through the preset release pressure, completing the transfer of a single rare cell. After the transfer is completed, control the Z-axis slide, planar slide, and multi-angle control console to return to their initial positions and clean the capillary glass microneedle. If it is necessary to pick other rare cells, repeat steps 3 to 5. Once the picking work is complete, turn off the power to the host computer and all modules, tidy up the experimental equipment and samples, and complete the entire automatic scanning and picking process.

[0014] The present invention discloses the following technical effects: The device is equipped with an automatic multicolor fluorescence scanning and recognition module, which, combined with a multi-channel fluorescence microscope and an image acquisition and analysis system, can achieve all-round scanning of samples without blind spots. Through image preprocessing and feature recognition algorithms, it can accurately distinguish between target rare cells and contaminating cells. At the same time, the planar slide moves the microscope at a constant speed, avoiding repeated scanning or omissions, greatly reducing human identification errors, improving identification efficiency and accuracy, and laying the foundation for subsequent picking operations.

[0015] The multi-angle control console can finely adjust the stage angle through rotation and dual pitch components to ensure that rare cells are in the optimal position for microneedle picking. The Z-axis slide of the micro-operation platform precisely controls the movement of the microneedle, and the adjustable pressure microinjector is preset with appropriate suction and release pressure to gently adsorb and release cells. Pressure is monitored in real time to avoid cell damage. At the same time, the nano-microcavity chip stably carries the sample, effectively ensuring the integrity and viability of the picked single cells.

[0016] The host computer coordinates and controls the collaborative operation of all modules. From device self-testing, scanning and identification, positioning and picking to cell transfer and device reset, the entire process is completed automatically without human intervention in core operations. This avoids the randomness and instability of manual operation, unifies operating parameters and procedures, standardizes single-cell operations of rare cells, and reduces the technical threshold and workload of operators. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the automatic scanning and picking device for single-cell manipulation of rare cells according to the present invention; Figure 2 This is a schematic diagram of the structure of the multi-angle control console of the present invention; Figure 3 This is a schematic diagram of the structure of the nanocavity chip of the present invention.

[0019] Among them, 1. Multichannel fluorescence microscope; 2. Planar sliding stage; 3. Z-axis sliding stage; 4. Capillary glass microneedle; 5. Nanoscale microcavity chip; 6. Multi-angle control console; 601. Base; 602. Rotating column; 603. First gear set; 604. First adjustment knob; 605. Mounting bracket; 606. Second gear set; 607. Second adjustment knob; 608. Pitch arm; 609. Third gear set; 610. Third adjustment knob; 611. Platform. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Reference Figures 1-3 This invention provides an automated scanning and picking device for single-cell manipulation of rare cells, comprising: An automatic multicolor fluorescence scanning and recognition module includes a multi-channel fluorescence microscope 1, a planar slide stage 2 mounted on the multi-channel fluorescence microscope 1, and the multi-channel fluorescence microscope 1 is connected to an image acquisition and analysis system. The multi-angle control console 6 includes a base 601, which is mounted on a flat slide 2. A rotation component, a first pitch component, and a second pitch component are mounted on the base 601. The first pitch component is mounted on the rotation component, and the second pitch component is mounted on the first pitch component. A stage 611 is mounted on the second pitch component. The micro-operation platform includes a Z-axis slide 3 mounted on a multi-channel fluorescence microscope 1. A capillary glass microneedle 4 is fixed at the end of the execution end of the Z-axis slide 3, and the rear end of the capillary glass microneedle 4 is connected to an adjustable pressure microinjector. The nano-microcavity chip 5 is placed on the stage 611. The host computer is used to control the operation of the entire device.

[0023] Further optimization of the design includes the following rotating components: Rotating column 602 is vertically rotatably connected to the top of base 601; The first gear set 603 is rotatably connected to the platform 611, and the rotating column 602 is fixed to the first gear set 603. The first adjustment knob 604 is rotatably connected to the stage 611, and the first adjustment knob 604 is fixed to one of the gears on the first gear set 603.

[0024] The core function of the rotating component is to enable 360° rotational adjustment of the stage 611, providing angular support for multi-angle observation and picking of rare cells. The rotating column 602 is vertically rotatably connected to the top of the stage 611, serving as the core actuator for rotational movement. The first gear set 603 is rotatably connected to the base 601, and the rotating column 602 is fixed to the first gear set 603, achieving synchronous movement between the gear set and the rotating column 602. When the first adjustment knob 604 is rotated, since the first adjustment knob 604 is fixed to a set of gears in the first gear set 603, the rotation of the knob will drive the gears to rotate, thereby driving the entire first gear set 603 to transmit power, ultimately causing the rotating column 602 fixed to the gear set to rotate around its own axis. This achieves rotational adjustment of the stage 611 and the nano-microcavity chip 5 above it, meeting the scanning and picking requirements at different angles.

[0025] In a further optimized design, the first pitch assembly includes a mounting bracket 605, which is rotatably connected to the top of the rotating column 602 via a mounting shaft. A second gear set 606 is mounted on the rotating column 602 via a fixing bracket. The second gear set 606 is fixed to the mounting shaft. A second adjustment knob 607 is rotatably connected to the fixing bracket. The second adjustment knob 607 is fixed to one set of gears in the second gear set 606.

[0026] The first pitch assembly is used to adjust the pitch of the stage 611 in the first direction, and works with the rotation assembly to achieve multi-angle attitude adjustment. The mounting bracket 605 is rotatably connected to the top of the rotating column 602 via a mounting shaft. The mounting bracket 605 can rotate around the mounting shaft to achieve pitch. The stage 611 is indirectly mounted on the mounting bracket 605 via subsequent components. The second gear set 606 mounted on the rotating column 602 via a fixing bracket is fixed to the mounting shaft to ensure that the gear set rotates synchronously with the mounting shaft. When the second adjustment knob 607 is rotated, the knob drives the corresponding gear in the second gear set 606 fixed to it to rotate. The gear set transmission drives the mounting shaft to rotate. The rotation of the mounting shaft drives the mounting bracket 605 to pitch around the mounting shaft, thereby enabling the stage 611 to achieve pitch angle adjustment in the first direction, ensuring precise alignment between the chip surface and the microscope objective.

[0027] In a further optimized design, the second pitch assembly includes a pitch boom 608, which is rotatably connected to a mounting frame 605. A platform 611 is mounted on the pitch boom 608. A third gear set 609 is mounted on the mounting frame 605 and is axially connected to the pitch boom 608. A third adjustment knob 610 is mounted on the mounting frame 605 and is fixed to one set of gears in the third gear set 609.

[0028] The second pitch assembly works in conjunction with the first pitch assembly to achieve pitch adjustment of the stage 611 in a second direction perpendicular to the first pitch direction, forming a dual pitch adjustment structure and improving the flexibility and accuracy of angle adjustment. The pitch arm 608 is rotatably connected to the mounting frame 605, and the stage 611 is directly mounted on the pitch arm 608. The rotation of the pitch arm 608 can directly drive the pitch of the stage 611. The third gear set 609 mounted on the mounting frame 605 is shaft-connected to the pitch arm 608 to achieve power transmission between the gear set and the pitch arm 608. When the third adjustment knob 610 is rotated, the knob drives the corresponding gear in the fixed third gear set 609 to rotate. The gear set drives the pitch arm 608 to rotate around its rotation axis with the mounting frame 605, thereby driving the stage 611 to achieve pitch adjustment in the second direction. This allows for precise fine-tuning of the position of rare cells, placing them in the optimal microneedle picking posture.

[0029] The scheme is further optimized. The nano-microcavity chip 5 includes a base layer and a top microfluidic channel layer. The base layer is detachably mounted on the planar slide 2. Several sets of microcavities are arranged in a two-dimensional array on the top surface of the base layer. An intermediate isolation layer is provided on the top surface of the base layer. Flow channels are arranged on the top microfluidic channel layer. Liquid inlet and liquid outlet are opened on the top microfluidic channel layer. The liquid inlet and liquid outlet are respectively connected to the flow channels, and the flow channels are connected to the microcavities.

[0030] The chip consists of a base layer, an intermediate isolation layer, and a top microfluidic layer. The base layer is detachably mounted on the planar slide 2 for easy chip replacement and cleaning. The two-dimensional array microcavities on the top surface of the base layer enable the individual separation and carrying of rare cells, preventing cell aggregation. Meanwhile, the intermediate isolation layer prevents cells from overflowing from the microcavities. The channels of the top microfluidic layer are connected to the microcavities. The inlet is used to inject samples containing rare cells, and the outlet is used to discharge excess sample or buffer solution. After the sample enters the channels through the inlet, it is evenly distributed into each two-dimensional array microcavity, achieving the dispersed carrying of rare cells and providing a stable sample carrier for subsequent automatic scanning identification and single-cell picking.

[0031] Further optimization of the design resulted in capillary glass microneedles with an inner diameter of 5–25 μm.

[0032] Further optimization of the scheme: the step resolution of the planar slide 2 in the XY direction is preferably no greater than 1μm.

[0033] Further optimization of the scheme resulted in the microcavities on the nano-microcavity chip 5 having a lateral dimension of 10–40 μm and a depth of 10–30 μm.

[0034] An automated scanning and picking method for single-cell manipulation of rare cells includes the following steps: Step 1: Turn on the host computer and start the entire device. Perform system self-checks on the automatic multicolor fluorescence scanning and recognition module, multi-angle control console 6, and micro-operation platform to ensure that each module is operating normally and the signal connection is stable. Drop the sample containing rare cells into the designated area of ​​the nano-microcavity chip 5, ensuring that the sample is evenly spread and does not overflow the chip area. Then, place the nano-microcavity chip 5 stably on the stage 611 of the multi-angle control console 6 and adjust the chip position to be in the center area of ​​the stage 611 for easy subsequent scanning and operation. At the same time, set the basic parameters of the multi-channel fluorescence microscope 1 through the host computer, adjust the adjustable pressure microinjector, remove air bubbles in the capillary glass microneedle 4 to ensure that the microneedle is unobstructed, preset the appropriate suction pressure and release pressure, and complete the device initialization and sample preparation. Step two: The host computer controls the rotation component, first pitch component, and second pitch component of the multi-angle control console 6 to drive the nanocavity chip 5 on the stage 611 to perform multi-angle adjustments, so that the chip surface is vertically aligned with the objective lens of the multi-channel fluorescence microscope 1 to ensure the clarity of the fluorescence scan. Then, the host computer controls the plane slide 2 to move the multi-channel fluorescence microscope 1. Based on the size of the nanocavity chip 5, the scanning area is set, and the scanning start and end points are clearly defined to avoid scanning omissions or redundant scans. At the same time, the focal length of the multi-channel fluorescence microscope 1 is adjusted to make the cell sample on the chip clearly imaged, preparing for subsequent fluorescence scanning and cell identification. Step 3: Activate the automatic multicolor fluorescence scanning and recognition module. The host computer controls the multi-channel fluorescence microscope 1 to perform an all-round automatic multicolor fluorescence scan on the sample on the nano-microcavity chip 5 according to the preset scanning path and parameters. The planar slide 2 drives the microscope to move at a uniform speed to ensure that there are no blind spots or repetitions in the scanning process. During the scanning process, the image acquisition and analysis system acquires cell images of each fluorescence channel in real time, preprocesses the acquired images to remove background noise and optimize image contrast, and then extracts cell contours through image recognition algorithms, calculates the average fluorescence intensity value of each fluorescence channel, and automatically identifies the target rare cells by combining the fluorescence characteristic parameters of rare cells. At the same time, the specific coordinate position of each rare cell on the nano-microcavity chip 5 is recorded, and the identification results and coordinate information are fed back to the host computer to complete the automatic identification and positioning of rare cells. Step four: Based on the identified rare cell coordinates, the host computer synchronously controls the planar slide 2 and the multi-angle control console 6 to move the target rare cell directly below the capillary glass microneedle 4 on the micromanipulation platform. Simultaneously, the angle of the stage 611 is finely adjusted via the rotation component, the first pitch component, and the second pitch component to ensure the rare cell is in the optimal picking position for the microneedle. Then, the Z-axis slide 3 of the micromanipulation platform is controlled to move downwards, causing the capillary glass microneedle 4 to slowly approach the target rare cell. When the microneedle tip reaches a preset distance, the host computer controls the adjustable pressure microinjector to activate the negative pressure suction function, gently adsorbing the target rare cell through the capillary glass microneedle 4. After adsorption, the Z-axis slide 3 is controlled to move upwards, causing the microneedle and cell to rise together, completing the picking operation of a single rare cell. During the picking process, the suction pressure is monitored in real time to prevent excessive pressure from damaging cell viability. Step 5: According to experimental requirements, the host computer controls the coordinated movement of the planar slide 2, the multi-angle control console 6, and the Z-axis slide 3 to move the capillary glass microneedle 4, which adsorbs rare cells, above the preset cell collection container. The position of the microneedle is adjusted to ensure that the tip of the microneedle is aligned with the designated area of ​​the collection container. Subsequently, the host computer controls the adjustable pressure microinjector to switch to positive pressure release mode, and the rare cells in the capillary glass microneedle 4 are smoothly released into the collection container through the preset release pressure, completing the transfer of a single rare cell. After the transfer is completed, the Z-axis slide 3, the planar slide 2, and the multi-angle control console 6 are controlled to return to their initial positions, and the capillary glass microneedle 4 is cleaned. If it is necessary to continue picking other rare cells, steps 3 to 5 are repeated. Once the picking work is completed, the power to the host computer and all modules is turned off, the experimental equipment and samples are organized, and the entire automatic scanning and picking process is completed.

[0035] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0036] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An automated scanning and picking device for single-cell manipulation of rare cells, characterized in that, include: An automatic multicolor fluorescence scanning and recognition module, comprising a multichannel fluorescence microscope (1), a planar slide stage (2) mounted on the multichannel fluorescence microscope (1), and the multichannel fluorescence microscope (1) being connected to an image acquisition and analysis system; A multi-angle control console (6) includes a base (601) mounted on the planar slide (2). A rotation component, a first pitch component, and a second pitch component are mounted on the base (601). The first pitch component is mounted on the rotation component, and the second pitch component is mounted on the first pitch component. A stage (611) is mounted on the second pitch component. The micro-operation platform includes a Z-axis slide (3) mounted on the multi-channel fluorescence microscope (1), and a capillary glass microneedle (4) is fixed at the end of the execution end of the Z-axis slide (3). The rear end of the capillary glass microneedle (4) is connected to an adjustable pressure microinjector. A nano-microcavity chip (5) is placed on the stage (611); The host computer is used to control the operation of the overall device.

2. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 1, characterized in that, The rotating component includes: A rotating column (602) is vertically rotatably connected to the top of the base (601); The first gear set (603) is rotatably connected to the platform (611), and the rotating column (602) is fixed on the first gear set (603); A first adjustment knob (604) is rotatably connected to the stage (611), and the first adjustment knob (604) is fixed to one of the gears on the first gear set (603).

3. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 2, characterized in that, The first pitch assembly includes a mounting bracket (605), which is rotatably connected to the top of the rotating column (602) via a mounting shaft. A second gear set (606) is mounted on the rotating column (602) via a fixing bracket. The second gear set (606) is fixed to the mounting shaft. A second adjustment knob (607) is rotatably connected to the fixing bracket. The second adjustment knob (607) is fixed to one set of gears in the second gear set (606).

4. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 3, characterized in that, The second pitch assembly includes a pitch boom (608) rotatably connected to the mounting frame (605), a platform (611) mounted on the pitch boom (608), a third gear set (609) mounted on the mounting frame (605), the third gear set (609) being axially connected to the pitch boom (608), and a third adjustment knob (610) mounted on the mounting frame (605), the third adjustment knob (610) being fixed to one set of gears in the third gear set (609).

5. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 1, characterized in that, The nano-microcavity chip (5) includes a base layer and a top microchannel layer. The base layer is detachably mounted on the planar slide (2). The top surface of the base layer is processed with several groups of microcavities arranged in a two-dimensional array. The top surface of the base layer is provided with an intermediate isolation layer. The top microchannel layer is provided with a flow channel. The top microchannel layer is provided with an inlet and an outlet. The inlet and the outlet are respectively connected to the flow channel. The flow channel is connected to the microcavity.

6. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 1, characterized in that, The capillary glass microneedles (4) have an inner diameter of 5–25 μm.

7. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 1, characterized in that, The step resolution of the planar slide (2) in the XY direction is preferably no greater than 1 μm.

8. The automatic scanning and picking device for single-cell manipulation of rare cells according to claim 5, characterized in that, The lateral dimension of the microcavity on the nanocavity chip (5) is 10–40 μm, and the depth is 10–30 μm.

9. An automated scanning and picking method for single-cell manipulation of rare cells, based on the automated scanning and picking device for single-cell manipulation of rare cells according to any one of claims 1-8, characterized in that, Includes the following steps: Step 1: Turn on the host computer and start the whole device through the host computer. Perform system self-check on the automatic multicolor fluorescence scanning and recognition module, multi-angle console (6), and micro-operation platform to ensure that each module is running normally and the signal connection is stable. Drop the sample containing rare cells into the designated area of ​​the nano-microcavity chip (5) to ensure that the sample is evenly spread and does not overflow the chip area. Then place the nano-microcavity chip (5) stably on the stage (611) of the multi-angle console (6) and adjust the chip position to be in the center area of ​​the stage (611) for easy subsequent scanning and operation. At the same time, set the basic parameters of the multi-channel fluorescence microscope (1) through the host computer, adjust the adjustable pressure microinjector, expel the air bubbles in the capillary glass microneedle (4) to ensure that the microneedle is unobstructed, preset the appropriate suction pressure and release pressure, and complete the device initialization and sample preparation work. Step 2: The rotation component, first pitch component and second pitch component of the multi-angle control console (6) are controlled by the host computer to drive the nano-microcavity chip (5) on the stage (611) to perform multi-angle adjustment so that the chip surface is vertically aligned with the objective lens of the multi-channel fluorescence microscope (1) to ensure the clarity of the fluorescence scan. Then, the planar slide (2) is controlled by the host computer to drive the multi-channel fluorescence microscope (1) to move. The scanning area is set according to the size of the nano-microcavity chip (5), and the scanning start and end points are clearly defined to avoid scanning omissions or redundant scans. At the same time, the focal length of the multi-channel fluorescence microscope (1) is adjusted so that the cell sample on the chip is clearly imaged, which is ready for subsequent fluorescence scanning and cell identification. Step 3: Start the automatic multicolor fluorescence scanning and recognition module. The host computer controls the multi-channel fluorescence microscope (1) to perform an all-round automatic multicolor fluorescence scan on the sample on the nano-microcavity chip (5) according to the preset scanning path and parameters. The planar slide (2) drives the microscope to move at a constant speed to ensure that there are no dead angles or repetitions in the scanning process. During the scanning process, the image acquisition and analysis system acquires cell images of each fluorescence channel in real time, preprocesses the acquired images to remove background noise and optimize image contrast, and then extracts cell outlines through image recognition algorithm, counts the average fluorescence intensity value of each fluorescence channel, and automatically identifies the target rare cells by combining the fluorescence characteristic parameters of rare cells. At the same time, the specific coordinate position of each rare cell on the nano-microcavity chip (5) is recorded, and the recognition results and coordinate information are fed back to the host computer to complete the automatic identification and positioning of rare cells. Step 4: Based on the identified rare cell coordinate information, the host computer synchronously controls the planar slide (2) and the multi-angle control console (6) to move the target rare cell directly below the capillary glass microneedle (4) of the micro-operation platform. At the same time, the angle of the stage (611) is finely adjusted by the rotation component, the first pitch component and the second pitch component to ensure that the rare cell is in the best picking position of the microneedle. Then, the Z-axis slide (3) of the micro-operation platform is controlled to move downward, driving the capillary glass microneedle (4) to slowly approach the target rare cell. When the tip of the microneedle reaches the preset distance, the host computer controls the adjustable pressure microinjector to start the negative pressure suction function. The target rare cell is gently adsorbed by the capillary glass microneedle (4). After the adsorption is completed, the Z-axis slide (3) is controlled to move upward, driving the microneedle and the cell to rise together to complete the picking operation of a single rare cell. The suction pressure is monitored in real time during the picking process to avoid excessive pressure from damaging cell activity. Step 5: According to the experimental requirements, the upper computer controls the coordinated movement of the planar slide (2), the multi-angle control console (6), and the Z-axis slide (3) to move the capillary glass microneedle (4) with adsorbed rare cells to the top of the preset cell collection container. Adjust the position of the microneedle to ensure that the tip of the microneedle is aligned with the designated area of ​​the collection container. Then, the upper computer controls the adjustable pressure microinjector to switch to positive pressure release mode. The rare cells in the capillary glass microneedle (4) are smoothly released into the collection container through the preset release pressure to complete the transfer of a single rare cell. After the transfer is completed, control the Z-axis slide (3), the planar slide (2), and the multi-angle control console (6) to return to the initial position and clean the capillary glass microneedle (4). If it is necessary to continue picking other rare cells, repeat steps 3 to 5. If the picking work is completed, turn off the power of the upper computer and each module, organize the experimental equipment and samples, and complete the entire automatic scanning and picking process.

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