Adherent cell injection method, system, device and medium based on double needle coordination

Abstract

The present invention discloses a method, system, device, and medium for injecting adherent cells based on double-needle coordination, which relates to the field of cell biology. The method comprises: obtaining scanned images of multiple adherent cells within a microscopic field of view; preprocessing the scanned images of the multiple adherent cells; applying a boundary tracking algorithm to the processed binary images; determining the number of adherent cells based on the outlines of the multiple adherent cells in the outline images of the multiple adherent cells; establishing a rectangular coordinate system on the outline images of the multiple adherent cells to determine the coordinates of each adherent cell and the injection point coordinates of each adherent cell; dividing the outline images of the multiple adherent cells into different injection areas based on the number of adherent cells and the coordinates of each adherent cell; applying a genetic algorithm to the injection point coordinates of each adherent cell in the different injection areas to determine an injection path; and injecting the adherent cells in each injection area according to the injection path. The present invention can improve the efficiency of adherent cell injection.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and in particular to a method, system, device and medium for injecting adherent cells based on double-needle coordination. Background Art

[0002] In recent decades, experimental methods that deliver exogenous substances into cells to study cellular pathology and function, such as viral-mediated, chemical-mediated, and physical-mediated methods, have attracted a growing number of researchers. Among these methods, microinjection, a physical-mediated method, has become the most mainstream method for cellular drug delivery due to its simplicity and the fact that it does not require the administration of drugs other than the injected exogenous substance.

[0003] Microinjection of adherent cells is a technique in which a glass microneedle is inserted into the adherent cells and a very small amount of exogenous substances is injected into the adherent cells, such as Figure 1 As shown. Traditionally, adherent cell microinjection is primarily performed manually by professionals. Because microinjection procedures are cumbersome, require significant manpower and resources, and are time-consuming, an increasing number of researchers have begun researching automated adherent cell microinjection techniques. For example, Viigipuu et al. developed an automated micromanipulator for adherent cell injection; Matsuoka et al. established a dual-micromanipulator microinjection system for rice protoplasts and mouse embryonic stem cells; Youoku et al. built a dual-camera microinjection system; Kallio et al. developed an impedance-based microinjection system; Becattini et al. achieved fully automated microinjection at a rate of 5.71 cells / minute; and Dong Sun et al. developed an automated microinjection system for adherent cell injection, achieving an injection rate of 25 cells / minute.

[0004] Therefore, the existing automated microinjection method only uses one injection microneedle for injection. Compared with the manual injection method, the microinjection process is not optimized and the injection efficiency cannot be greatly improved. Summary of the Invention

[0005] The purpose of the present invention is to provide a method, system, device and medium for adherent cell injection based on double needle coordination, which can improve the efficiency of adherent cell injection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] A method for injecting adherent cells based on double-needle coordination, the method comprising:

[0008] Acquire scanning images of multiple adherent cells within the microscopic field of view;

[0009] preprocessing the scanned images of the plurality of adherent cells to obtain processed binary images;

[0010] applying a boundary tracking algorithm to the processed binary image to determine contour images of a plurality of adherent cells;

[0011] determining the number of adherent cells according to the outlines of the plurality of adherent cells in the outline image of the plurality of adherent cells;

[0012] establishing a rectangular coordinate system on the contour images of the plurality of adherent cells, and determining the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell;

[0013] Dividing the contour images of the plurality of adherent cells into different injection areas according to the number of the adherent cells and the coordinates of each adherent cell; the injection areas include a first injection area and a second injection area;

[0014] applying a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine an injection path;

[0015] According to the injection route, the adherent cells in each injection area are injected.

[0016] Optionally, preprocessing the scanned images of the plurality of adherent cells to obtain a processed binary image specifically includes:

[0017] converting the scanned images of the plurality of adherent cells into grayscale images;

[0018] performing binarization processing on the grayscale image to determine a binarized image;

[0019] Applying median filtering to remove noise from the binary image to determine a denoised binary image;

[0020] Performing a closing operation of dilation and erosion on the denoised binary image to obtain a processed binary image.

[0021] Optionally, establishing a rectangular coordinate system on the contour images of the plurality of adherent cells to determine the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell specifically includes:

[0022] determining the coordinates of the pixel points on the outline of each adherent cell and the radius of each adherent cell according to the rectangular coordinate system;

[0023] According to the coordinates of the pixel points, the coordinates of the pixel point with the smallest ordinate on the outline of each adherent cell are determined to obtain the coordinates of each adherent cell;

[0024] The ordinate of each adherent cell coordinate is added to the radius of each adherent cell to obtain the ordinate of the injection point coordinate of each adherent cell;

[0025] The injection point coordinates of the plurality of adherent cells are obtained according to the abscissa of the coordinates of each adherent cell and the ordinate of the injection point coordinates of each adherent cell.

[0026] Optionally, dividing the contour images of the plurality of adherent cells into different injection areas according to the number of the adherent cells and the coordinates of each adherent cell specifically includes:

[0027] determining whether the number of the adherent cells is an odd number or an even number;

[0028] When the number of the adherent cells is an even number, dividing the contour images of the plurality of adherent cells into a first injection area and a second injection area on an even basis according to the size of the ordinate in the coordinates of each adherent cell;

[0029] When the number of the adherent cells is an odd number, the number of the adherent cells is 2N+1, and from the coordinates of the adherent cells sorted from large to small according to the vertical coordinate, the first N adherent cells are selected as the first injection area, and the last N adherent cells are selected as the second injection area; when the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the Nth adherent cell coordinate is less than the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the N+2th adherent cell coordinate, the N+1th adherent cell belongs to the first injection area; otherwise, the N+1th adherent cell belongs to the second injection area.

[0030] Optionally, the method further includes:

[0031] Determine whether multiple adherent cells within the current microscopic field of view have been injected;

[0032] When the injection is complete, switch to the next microscopic field;

[0033] When the injection is not completed, the injection is continued for the plurality of adherent cells within the current microscopic field of view.

[0034] Optionally, injecting the adherent cells in each injection area according to the injection path specifically includes:

[0035] During injection, the first injection needle starts injecting from the starting point of the corresponding injection path at a first set angle within the first injection area, while the second injection needle starts injecting from the starting point of the corresponding injection path at a second set angle within the second injection area; the first injection needle and the second injection needle do not contact each other during the injection process;

[0036] After the first injection needle completes the injection of the current adherent cell in the first injection area, the needle tip is raised to a first set height, and the next adherent cell is injected according to the injection path corresponding to the first injection area, until the injection of all adherent cells in the first injection area is completed;

[0037] After the second injection needle completes the injection of the current adherent cells in the second injection area, the needle tip is lifted upward to a second set height, and the next adherent cell is injected according to the injection path corresponding to the second injection area until the injection of all adherent cells in the second injection area is completed.

[0038] A double-needle coordinated adherent cell injection system is applied to the above-mentioned double-needle coordinated adherent cell injection method, the system comprising:

[0039] an acquisition module, used for acquiring scanning images of multiple adherent cells within a microscopic field of view;

[0040] a preprocessing module, configured to preprocess the scanned images of the plurality of adherent cells to obtain a processed binary image;

[0041] a contour determination module, configured to apply a boundary tracking algorithm to the processed binary image to determine contour images of a plurality of adherent cells;

[0042] a number determination module, configured to determine the number of adherent cells based on the outlines of the plurality of adherent cells in the outline image of the plurality of adherent cells;

[0043] a coordinate determination module, configured to establish a rectangular coordinate system on the contour images of the plurality of adherent cells, and determine the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell;

[0044] a region division module, configured to divide the contour image of the plurality of adherent cells into different injection regions according to the number of the adherent cells and the coordinates of each adherent cell; the injection regions include a first injection region and a second injection region;

[0045] a path determination module, configured to apply a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine an injection path;

[0046] The injection module is used to inject the adherent cells in each injection area according to the injection path.

[0047] Optionally, the preprocessing module includes:

[0048] a conversion submodule, configured to convert the scanned images of the plurality of adherent cells into grayscale images;

[0049] A binarization image submodule is used to perform binarization processing on the grayscale image to determine a binarized image;

[0050] a denoising submodule, configured to remove noise from the binary image by applying a median filter, and determine a denoised binary image;

[0051] The dilation and erosion submodule is used to perform dilation and erosion closing operations on the denoised binary image to obtain a processed binary image.

[0052] An electronic device includes a memory and a processor, wherein the memory is used to store a computer program, and the processor runs the computer program to enable the electronic device to perform the above-mentioned double-needle coordinated adherent cell injection method.

[0053] A computer-readable storage medium stores a computer program, which, when executed by a processor, implements the above-mentioned adherent cell injection method based on double-needle coordination.

[0054] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0055] The present invention provides a method for injecting adherent cells based on double needle coordination, comprising: obtaining scanning images of multiple adherent cells within a microscopic field of view; preprocessing the scanning images of the multiple adherent cells to obtain processed binary images; applying a boundary tracking algorithm to the processed binary images to determine contour images of the multiple adherent cells; determining the number of adherent cells based on the contours of the multiple adherent cells in the contour images of the multiple adherent cells; establishing a rectangular coordinate system on the contour images of the multiple adherent cells to determine the coordinates of each adherent cell and the injection point coordinates of each adherent cell; dividing the contour images of the multiple adherent cells into different injection areas based on the number of adherent cells and the coordinates of each adherent cell; the injection areas include a first injection area and a second injection area; applying a genetic algorithm to the injection point coordinates of each adherent cell in the different injection areas to determine an injection path; and injecting the adherent cells in each injection area based on the injection path. The present invention achieves the purpose of improving injection efficiency by setting an adherent cell injection process based on double needle coordination, and completing the injection process by identifying adherent cells, planning the injection path, and coordinating the operation of the double needles, thereby realizing the double needle coordinated adherent cell injection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0057] Figure 1 Figure 1 is a diagram of adherent cell microinjection;

[0058] Figure 2 A flow chart of the adherent cell injection method based on double-needle coordination provided by the present invention;

[0059] Figure 3 An injection flow chart of a specific embodiment provided by the present invention;

[0060] Figure 4 Schematic diagram of the double-needle coordinated injection experimental system provided by the present invention;

[0061] Figure 5 A flow chart of cell identification provided by the present invention;

[0062] Figure 6 A schematic diagram of a cell recognition image provided by the present invention;

[0063] Figure 7 A schematic diagram of the four neighborhood directions provided by the present invention;

[0064] Figure 8 A schematic diagram of the eight neighborhood directions provided by the present invention;

[0065] Figure 9 This is a schematic diagram of the cell identification source image provided by the present invention;

[0066] Figure 10 This is a schematic diagram of the cell recognition simulation result image provided by the present invention;

[0067] Figure 11 This is a schematic diagram of the path planning image provided by the present invention;

[0068] Figure 12 A schematic diagram of double needle coordination provided by the present invention;

[0069] Figure 13 This is a module diagram of the adherent cell injection system based on double needle coordination provided by the present invention.

[0070] Explanation of symbols:

[0071] 1- Acquisition module, 2- Preprocessing module, 3- Contour determination module, 4- Quantity determination module, 5- Coordinate determination module, 6- Area division module, 7- Path determination module, 8- Injection module. DETAILED DESCRIPTION

[0072] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0073] The purpose of the present invention is to provide a method, system, device and medium for adherent cell injection based on double needle coordination, which can improve the efficiency of adherent cell injection.

[0074] Existing microinjection methods use only one microneedle in a microinjection system to inject adherent cells. However, adherent cells do not require a gripper to hold the cells in place during microinjection. If two microneedles could be used in tandem to perform automated microinjection of adherent cells, injection efficiency would be significantly improved compared to using only one microneedle. Ideally, this efficiency could even be doubled. Therefore, research into automated adherent cell injection methods using coordinated dual-needle technology is crucial. Therefore, the present invention proposes an automated adherent cell injection method based on double-needle coordination. First, an adherent cell injection process based on double-needle coordination is designed; secondly, key technologies in the automatic adherent cell injection based on double-needle coordination are solved, including adherent cell recognition, injection path planning and double-needle coordinated operation; finally, an adherent cell microinjection experiment based on double-needle coordination is carried out. The experimental results show that the injection speed of this method can reach 35 cells / minute, which is 75% higher than the manual injection method using a single needle (20 cells / minute) and 40% higher than the automated adherent cell injection method using a single needle (25 cells / minute).

[0075] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0076] like Figure 2 and Figure 3 As shown, the present invention provides a method for injecting adherent cells based on double needle coordination, the method comprising:

[0077] Step S1: Acquire scanning images of multiple adherent cells within the microscopic field of view.

[0078] In practical applications, taking HumanEmbryonic Kidney 293Cells (HEK293 cells, human embryonic kidney cells) as an example, HumanEmbryonic Kidney 293Cells (HEK293 cells, human embryonic kidney cells) are first processed as follows: (1) The cells are evenly spread in a 35mm culture dish containing complete culture medium (high-glucose DMEM containing 10% fetal bovine serum, 1% penicillin and streptomycin) and cultured in an incubator at 37°C and 5% CO2 concentration. (2) The culture medium of the experimental cells is discarded in the clean bench and the cells are rinsed with sterile PBS buffer; after discarding the PBS buffer, 200μL trypsin is added and the cells are allowed to stand at room temperature for 1 minute for digestion. (3) 1mL of complete culture medium is added to terminate the digestion, and the cells are gently blown with a pipette to slowly detach the cells from the culture dish to form a cell suspension. (4) The cell suspension is transferred into a centrifuge tube and centrifuged at 2000 rpm for 4 minutes. After centrifugation, discard the supernatant of the cells obtained, add 9-10 ml of complete culture medium, shake the culture dish gently to evenly distribute the cells, and then place the cells in an incubator and culture them until they grow adherently.

[0079] Experimental setup such as Figure 4 As shown, it includes a standard inverted microscope (Eclipse TI-E, Nikon, Japan); a high-speed industrial camera (aca640-120gm / gc, Basler, Germany); a motorized stage (ProScan III, Prior, UK); two beveled micropipette (inner diameter approximately 1-2 μm) connected to a pneumatic microinjector (XenoWorks BRE, Sutter, USA) for injection; two three-degree-of-freedom motorized micromanipulator arms (MP285, Sutter, USA) to control the position of the micropipette; a needle holder (HI-7, Narishige, Japan, equipped with a micropipette with a 30° tilt angle); and an industrial computer for controlling multiple motion control devices and real-time image processing.

[0080] The cultured adherent cells were scanned and the current field of view was retrieved as input, and the positions of the two injection needle tips were manually marked.

[0081] Step S2: Pre-process the scanned images of the plurality of adherent cells to obtain a processed binary image. Figure 5 and Figure 6 As shown, Figure 6 Part (a) to Figure 6 Part (f) of the paper gives the cell recognition process. Figure 6 Part (a) is a microscopic image. Figure 6Part (b) is the converted binary image. Figure 6 Part (c) is the image after median filtering. Figure 6 Part (d) is the image after dilation processing. Figure 6 Part (e) is the image after corrosion processing. Figure 6 Part (f) shows the image after contour extraction. Perform morphological operations on the image in the field of view to identify all cells, draw their outlines, and count the adherent cells. Scan the microscope field of view to the front end. It is important to keep the experimental environment's brightness as stable as possible; large fluctuations can affect the experimental results.

[0082] S2 specifically includes:

[0083] Step S21: converting the scanned images of the plurality of adherent cells into grayscale images.

[0084] Step S22: performing binarization processing on the grayscale image to determine a binarized image.

[0085] Step S23: applying median filtering to remove noise from the binary image, and determining a denoised binary image.

[0086] Step S24: performing dilation and erosion closing operations on the denoised binary image to obtain a processed binary image.

[0087] Step S3: applying a boundary tracking algorithm to the processed binary image to determine the contour images of the plurality of adherent cells.

[0088] Specifically, the boundary tracking algorithm is used to detect the boundaries of a binary image, that is, to find the outline and edge of a graphic. The specific implementation process is as follows:

[0089] Step S31: Scan the microscopic field of view from left to right and from top to bottom to find the boundary point (pixel) P0 at the upper left of the cell to be detected. Define a variable temp to store the moving direction of the boundary point. There are two ways to detect the boundary, as follows:

[0090] The first one: When detecting the boundary in the 4-field method, such as Figure 7 As shown, initialize temp=3.

[0091] The second type: When detecting boundaries in the 8-field method, such as Figure 8 As shown, initialize temp=3.

[0092] Step S32: When the initial boundary point P0 is scanned, a 3*3 area centered on the current pixel P0 is searched in a counterclockwise direction. Depending on the boundary detection method, the corresponding search method is:

[0093] When detecting the boundary using the 4-field method, search in the Res direction and execute Res = (temp + 3) mod 4. When detecting the boundary using the 8-field method, search in the Res direction and execute:

[0094] Res=(temp+7)mod8, when temp is an even number.

[0095] Res=(temp+6)mod8, when temp is an odd number.

[0096] Step S33: When a new cell boundary is encountered (the first pixel found with the same value as the current pixel is a new cell boundary element Pn), update temp.

[0097] Step S34: If the current cell boundary element Pn is equal to the second boundary element P1, and the previous cell boundary element Pn-1 is equal to P0, then stop. Otherwise, repeat step 32.

[0098] The final detected intracellular boundary is composed of a series of point sets of pixels P0, P1, ..., Pn-2.

[0099] In practical applications, such as Figure 9 and Figure 10 As shown. The cell recognition program of the present invention identified the outlines of 13 cells in the source image. It should be noted that due to differences in experimental environment and cell types, the brightness of cells in the field of view may vary. This requires adjusting the parameters of the function to maximize the accuracy and clarity of the detection results. The parameters used in the present invention are as follows:

[0100] (1) Binarization cvThreshold(constArr*src,CvArr*dst,double threshold,doublemax_value,intthreshold_type): the pixel threshold is 160, 255.

[0101] (2) Median filtering void medianBlur(InputArray src, OutputArray dst, int ksize) where the filter radius is 7.

[0102] (3) Erosion and dilation void dilate(InputArray src, OutputArray dst, InputArraykernel), void erode(InputArray src, OutputArray dst, InputArraykernel): The area of ​​erosion and dilation is 15*15.

[0103] Step S4: determining the number of adherent cells according to the contours of the plurality of adherent cells in the contour image of the plurality of adherent cells.

[0104] Step S5: establishing a rectangular coordinate system on the contour images of the plurality of adherent cells, and determining the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell.

[0105] As a specific embodiment, an XYZ rectangular coordinate system is established, with the plane of the adherent cells as the XY plane and the direction of the injection needle away from the XY plane as the Z axis. The coordinates of the lower endpoint of each adherent cell are calculated and used as the coordinates of the cell. During injection, the injection point coordinates are set to the coordinates of the cell's lower endpoint minus the cell radius R, so that the injection point is approximately at the morphological center of the adherent cell.

[0106] S5 specifically includes:

[0107] Step S51: determining the coordinates of the pixel points on the outline of each adherent cell and the radius of each adherent cell according to the rectangular coordinate system.

[0108] Step S52: According to the coordinates of the pixel points, the coordinates of the pixel point with the smallest ordinate on the outline of each adherent cell are determined to obtain the coordinates of each adherent cell.

[0109] Step S53: Adding the ordinate of each adherent cell coordinate to the radius of the corresponding adherent cell to obtain the ordinate of the injection point coordinate of each adherent cell.

[0110] Step S54: obtaining the injection point coordinates of the plurality of adherent cells according to the abscissa of the coordinates of each adherent cell and the ordinate of the injection point coordinates of each adherent cell.

[0111] Step S6: dividing the contour images of the plurality of adherent cells into different injection areas according to the number of the adherent cells and the coordinates of each adherent cell; the injection areas include a first injection area and a second injection area.

[0112] Specifically, it is determined whether the number of the adherent cells is an odd number or an even number.

[0113] When the number of the adherent cells is an even number, the contour images of the plurality of adherent cells are evenly divided into a first injection area and a second injection area according to the size of the ordinate in the coordinates of each adherent cell.

[0114] When the number of the adherent cells is an odd number, the number of the adherent cells is 2N+1, and from the coordinates of the adherent cells sorted from large to small according to the vertical coordinate, the first N adherent cells are selected as the first injection area, and the last N adherent cells are selected as the second injection area; when the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the Nth adherent cell coordinate is less than the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the N+2th adherent cell coordinate, the N+1th adherent cell belongs to the first injection area; otherwise, the N+1th adherent cell belongs to the second injection area.

[0115] In practical applications, all adherent cells are divided into upper and lower parts according to the size of the vertical coordinate. If the total number of cells is even, the upper and lower parts are divided equally. If the total number of cells is odd, then when the vertical coordinate of the cell in the middle is close to the lowest cell in the upper part, the cell is placed in the upper part for injection. When the vertical coordinate of the cell in the middle is closest to the highest cell in the lower part, the cell is placed in the lower part for injection.

[0116] Step S7: applying a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine the injection path.

[0117] As a specific implementation method, a genetic algorithm is used to plan the path for the upper and lower sections of adherent cells, and then two shortest injection paths are obtained. The genetic algorithm is a process that continuously optimizes the path length through thousands of iterations. Therefore, it is difficult to further reduce the length of the path planned by the genetic algorithm. The injection path obtained is as follows Figure 11 shown.

[0118] Step S8: injecting the adherent cells in each injection area according to the injection path.

[0119] As a specific embodiment, when performing an injection, the first injection needle starts injecting from the starting point of the corresponding injection path at a first set angle within the first injection area, while the second injection needle starts injecting from the starting point of the corresponding injection path at a second set angle within the second injection area; the first injection needle and the second injection needle have no contact during the injection process.

[0120] After the first injection needle completes the injection of the current adherent cells in the first injection area, the needle tip is lifted upward to a first set height, and the next adherent cell is injected according to the injection path corresponding to the first injection area until the injection of all adherent cells in the first injection area is completed.

[0121] After the second injection needle completes the injection of the current adherent cells in the second injection area, the needle tip is lifted upward to a second set height, and the next adherent cell is injected according to the injection path corresponding to the second injection area until the injection of all adherent cells in the second injection area is completed.

[0122] In practical applications, PBS was injected to verify the system's performance. In the experiment, cells were injected using a Sutter syringe, with the compensation pressure set to 30 hPa. The injection pressure was increased in 50-hPa intervals from 300 to 800 hPa to find the optimal injection pressure. The experiment found that an injection pressure between 400 and 600 hPa provided an appropriate injection volume, and cells adhered well to the injection wall. Furthermore, after injection, the cells were observed to fluctuate within a certain range centered on the injection point, which then returned to their original state. The adherent cell injection process was controlled by two manipulators, one on each side. The position of the microneedle tip was stored by clicking the mouse in the microscopic image. The program's cell recognition function then acquired the cell coordinates, linking the needle tip and cell positions in real time for position acquisition and control. Once the cell coordinates were acquired, the two manipulators coordinated their movements and controlled the syringe to inject PBS, achieving rapid cell injection.

[0123] like Figure 12 As shown, the left injection needle is used to inject the upper half of the adherent cells, and the right injection needle is used to inject the lower half of the adherent cells. Each needle is moved to 10 μm above and to the right of the target cell, then inserted into the cell at a 30-degree angle downward. The drug is injected using an air pump to complete the injection of one adherent cell. Each needle is then raised 10 μm and injected into the next adherent cell along the planned path. Following the injection path, the upper and lower half of the adherent cells are injected.

[0124] In addition, the double-needle coordinated adherent cell injection method provided by the present invention further includes:

[0125] Determine whether the injection of multiple adherent cells in the current microscopic field of view is complete; when the injection is complete, switch to the next microscopic field of view; when the injection is not complete, continue to inject the multiple adherent cells in the current microscopic field of view.

[0126] As a specific implementation, it is determined whether all adherent cells within the current microscopic field of view have been injected. If the injection is completed, the stage is moved to switch to the next microscopic field of view, and the above steps S1 to S8 are repeated.

[0127] In practical applications, the operation process of the double-needle coordinated adherent cell injection method provided by the present invention is as follows:

[0128] (1) Find the appropriate cell in the field of view and adjust the focal plane to the cell plane (i.e., bottom surface).

[0129] (2) Adjust the injection needle to the field of view and lower it to the bottom in the Z direction.

[0130] (3) Store the current positions of the two needle tips. Right-click the right needle tip in the field of view to obtain the pixel coordinates of the right needle tip. Click the left needle tip again to obtain the coordinates of the left needle tip.

[0131] (4) Automatically identify the positions of all cells in the field of view, divide the cells into two parts, and perform path planning for these two parts separately.

[0132] (5) Start injection: two injection needles are inserted obliquely downward to inject liquid, the injection needle is lifted and moved to the next cell, and the process is repeated until all selected cells are injected. During the process of the injection needle inserting obliquely downward, the Z-direction movement distance_z is 10 μm, and the X-direction movement distance_x = distance_z / tanα, where α is the angle between the injection needle and the horizontal plane, which is set to 30 degrees in this experiment.

[0133] (6) Move the stage to the next cell field of view and repeat the whole process.

[0134] In this experiment, 70 adherent cells were microinjected in two minutes, achieving an injection rate of 35 cells per minute. This represents a 75% increase over the manual injection rate of 20 cells per minute using a single needle and a 40% increase over the automated injection rate of 25 cells per minute using a single needle.

[0135] Example 2

[0136] In order to execute the method corresponding to the above embodiment 1 and achieve the corresponding functions and technical effects, a double-needle coordinated adherent cell injection system is provided below. Figure 13 As shown, the system includes:

[0137] The acquisition module 1 is used to acquire scanning images of multiple adherent cells within the microscopic field of view.

[0138] The preprocessing module 2 is configured to preprocess the scanned images of the plurality of adherent cells to obtain processed binary images.

[0139] The contour determination module 3 is configured to apply a boundary tracking algorithm to the processed binary image to determine contour images of a plurality of adherent cells.

[0140] The number determining module 4 is configured to determine the number of adherent cells according to the contours of the plurality of adherent cells in the contour image of the plurality of adherent cells.

[0141] The coordinate determination module 5 is used to establish a rectangular coordinate system on the contour images of the plurality of adherent cells, and determine the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell.

[0142] The region division module 6 is configured to divide the contour image of the plurality of adherent cells into different injection regions according to the number of the adherent cells and the coordinates of each adherent cell; the injection regions include a first injection region and a second injection region.

[0143] The path determination module 7 is configured to apply a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine the injection path.

[0144] The injection module 8 is used to inject the adherent cells in each injection area according to the injection path.

[0145] Wherein, the preprocessing module 2 includes:

[0146] The conversion submodule is used to convert the scanned images of the plurality of adherent cells into grayscale images.

[0147] The binarization image submodule is used to perform binarization processing on the grayscale image to determine a binarized image.

[0148] The denoising submodule is used to remove noise from the binary image by applying a median filter to determine a denoised binary image.

[0149] The dilation and erosion submodule is used to perform dilation and erosion closing operations on the denoised binary image to obtain a processed binary image.

[0150] Example 3

[0151] An embodiment of the present invention provides an electronic device, including a memory and a processor, wherein the memory is used to store a computer program, and the processor runs the computer program to enable the electronic device to perform the double-needle coordinated adherent cell injection method of embodiment 1.

[0152] Optionally, the above-mentioned electronic device may be a server.

[0153] In addition, an embodiment of the present invention further provides a computer-readable storage medium storing a computer program, which, when executed by a processor, implements the adherent cell injection method based on double-needle coordination of the first embodiment.

[0154] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the systems disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method description.

[0155] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only intended to help understand the method and core concept of the present invention. At the same time, those skilled in the art will find that the specific implementation methods and application scopes may vary based on the concept of the present invention. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims

1. A method for injecting adherent cells based on double-needle coordination, characterized in that: The method comprises: Acquire scanning images of multiple adherent cells within the microscopic field of view; preprocessing the scanned images of the plurality of adherent cells to obtain processed binary images; applying a boundary tracking algorithm to the processed binary image to determine contour images of a plurality of adherent cells; determining the number of adherent cells according to the outlines of the plurality of adherent cells in the outline image of the plurality of adherent cells; establishing a rectangular coordinate system on the contour images of the plurality of adherent cells, and determining the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell; Dividing the contour images of the plurality of adherent cells into different injection areas according to the number of the adherent cells and the coordinates of each adherent cell; the injection areas include a first injection area and a second injection area; applying a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine an injection path; According to the injection route, the adherent cells in each injection area are injected.

2. The method for injecting adherent cells based on double needle coordination according to claim 1, characterized in that: The preprocessing of the scanned images of the plurality of adherent cells to obtain a processed binary image specifically includes: converting the scanned images of the plurality of adherent cells into grayscale images; performing binarization processing on the grayscale image to determine a binarized image; Applying median filtering to remove noise from the binary image to determine a denoised binary image; Performing a closing operation of dilation and erosion on the denoised binary image to obtain a processed binary image.

3. The method for injecting adherent cells based on double needle coordination according to claim 1, characterized in that: The step of establishing a rectangular coordinate system on the contour images of the plurality of adherent cells and determining the coordinates of each adherent cell and the injection point coordinates of each adherent cell specifically includes: determining the coordinates of the pixel points on the outline of each adherent cell and the radius of each adherent cell according to the rectangular coordinate system; According to the coordinates of the pixel points, the coordinates of the pixel point with the smallest ordinate on the outline of each adherent cell are determined to obtain the coordinates of each adherent cell; The ordinate of each adherent cell coordinate is added to the radius of each adherent cell to obtain the ordinate of the injection point coordinate of each adherent cell; The injection point coordinates of the plurality of adherent cells are obtained according to the abscissa of the coordinates of each adherent cell and the ordinate of the injection point coordinates of each adherent cell.

4. The method for injecting adherent cells based on double needle coordination according to claim 1, characterized in that: The step of dividing the contour images of the plurality of adherent cells into different injection areas according to the number of the adherent cells and the coordinates of each adherent cell comprises: determining whether the number of the adherent cells is an odd number or an even number; When the number of the adherent cells is an even number, dividing the contour images of the plurality of adherent cells into a first injection area and a second injection area on an even basis according to the size of the ordinate in the coordinates of each adherent cell; When the number of the adherent cells is an odd number, the number of the adherent cells is 2N+1, and from the coordinates of the adherent cells sorted from large to small according to the vertical coordinate, the first N adherent cells are selected as the first injection area, and the last N adherent cells are selected as the second injection area; when the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the Nth adherent cell coordinate is less than the difference between the vertical coordinate of the N+1th adherent cell coordinate and the vertical coordinate of the N+2th adherent cell coordinate, the N+1th adherent cell belongs to the first injection area; otherwise, the N+1th adherent cell belongs to the second injection area.

5. The method for injecting adherent cells based on double needle coordination according to claim 1, characterized in that: The method further comprises: Determine whether multiple adherent cells within the current microscopic field of view have been injected; When the injection is complete, switch to the next microscopic field; When the injection is not completed, the injection is continued for the plurality of adherent cells within the current microscopic field of view.

6. The method for injecting adherent cells based on double needle coordination according to claim 1, characterized in that: Injecting the adherent cells in each injection area according to the injection path specifically includes: During injection, the first injection needle starts injecting from the starting point of the corresponding injection path at a first set angle within the first injection area, while the second injection needle starts injecting from the starting point of the corresponding injection path at a second set angle within the second injection area; the first injection needle and the second injection needle do not contact each other during the injection process; After the first injection needle completes the injection of the current adherent cell in the first injection area, the needle tip is raised to a first set height, and the next adherent cell is injected according to the injection path corresponding to the first injection area, until the injection of all adherent cells in the first injection area is completed; After the second injection needle completes the injection of the current adherent cells in the second injection area, the needle tip is lifted upward to a second set height, and the next adherent cell is injected according to the injection path corresponding to the second injection area until the injection of all adherent cells in the second injection area is completed.

7. A double-needle coordinated adherent cell injection system, characterized in that: The system comprises: an acquisition module, used for acquiring scanning images of multiple adherent cells within a microscopic field of view; a preprocessing module, configured to preprocess the scanned images of the plurality of adherent cells to obtain a processed binary image; a contour determination module, configured to apply a boundary tracking algorithm to the processed binary image to determine contour images of a plurality of adherent cells; a number determination module, configured to determine the number of adherent cells based on the outlines of the plurality of adherent cells in the outline image of the plurality of adherent cells; a coordinate determination module, configured to establish a rectangular coordinate system on the contour images of the plurality of adherent cells, and determine the coordinates of each adherent cell and the coordinates of the injection point of each adherent cell; a region division module, configured to divide the contour image of the plurality of adherent cells into different injection regions according to the number of the adherent cells and the coordinates of each adherent cell; the injection regions include a first injection region and a second injection region; a path determination module, configured to apply a genetic algorithm to the injection point coordinates of each adherent cell within the different injection areas to determine an injection path; The injection module is used to inject the adherent cells in each injection area according to the injection path.

8. The double-needle coordinated adherent cell injection system according to claim 7, characterized in that: The pre-processing module comprises: a conversion submodule, configured to convert the scanned images of the plurality of adherent cells into grayscale images; A binarization image submodule is used to perform binarization processing on the grayscale image to determine a binarized image; a denoising submodule, configured to remove noise from the binary image by applying a median filter, and determine a denoised binary image; The dilation and erosion submodule is used to perform dilation and erosion closing operations on the denoised binary image to obtain a processed binary image.

9. An electronic device, characterized in that: The electronic device comprises a memory and a processor, wherein the memory is used to store a computer program, and the processor runs the computer program to enable the electronic device to perform the adherent cell injection method based on double-needle coordination according to any one of claims 1 to 6.

10. A computer-readable storage medium, characterized in that The device stores a computer program, which, when executed by a processor, implements the adherent cell injection method based on double-needle coordination as claimed in any one of claims 1 to 6.

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