A method for precise extraction of cell polar bodies

Abstract

This invention provides a precise method for extracting cell polar bodies, belonging to the field of subcellular micromanipulation technology. The method includes the following steps: S1: Subtracting positive and negative defocused images, binarizing them, and using polar coordinate information to locate the polar body; S2: Performing force analysis on the polar body, and determining the motion trajectory of the microneedle approaching the polar body through finite element simulation modeling; S3: Inserting the microneedle into the cell, using an active disturbance rejection controller motion control algorithm to compensate for disturbances and errors during the microneedle's movement, correcting the microneedle's motion trajectory, and ensuring the microneedle approaches the polar body along the trajectory determined in S2; the microneedle then extracts the polar body. This invention manipulates cell polar bodies under a microscope while the microneedle is fixed in a suction-holding state. It allows the microneedle to be moved to a suitable position for polar body removal without staining the polar body, avoiding the difficulties caused by photobleaching and fluorescence fields resulting from staining, which can affect microneedle movement and the calculation of cytoplasm removal. It also reduces the damage to oocyte viability caused by excessive cytoplasm removal.

Description

Technical Field

[0001] This invention belongs to the field of subcellular micromanipulation technology, and specifically relates to a method for the precise extraction of cell polar bodies. Background Technology

[0002] As a product of mitosis in oocytes, the polar body contains a genetic backup completely identical to the cell nucleus. Polar body biopsy, a genetic test performed on the polar body, can identify genetic defects in oocytes, preventing the birth of genetically defective fetuses. A key step in polar body biopsy is controlling the microneedle's entry into the cell to extract the polar body. This process requires minimizing the amount of cytoplasm aspirated, as the scattered nuclear genetic material in the cytoplasm can interfere with the biopsy results, while excessive cytoplasm loss can affect the subsequent development of the oocyte. Therefore, precise manipulation of the polar body is essential during polar body biopsy. Because the polar body is relatively small compared to the cytoplasm and is essentially transparent, it is difficult to accurately locate using conventional image processing algorithms. Furthermore, the compression caused by cell deformation during microneedle movement within the cell can cause the polar body to drift, making it difficult to control the microneedle's approach to the polar body for extraction. Summary of the Invention

[0003] This invention addresses the technical problems existing in the prior art by providing a precise method for extracting cell polar bodies. It employs defocus imaging to increase the contrast between the polar body and the background, uses polar coordinate information to distinguish the polar body from the background, completes the polar body positioning, determines the required trajectory of the microneedle to approach the polar body within the cell through force analysis combined with finite element modeling, and uses ADRC to correct the microneedle's movement trajectory to approach the polar body and complete subsequent operations.

[0004] The technical solution adopted in this invention is: a method for precise extraction of cell polar bodies, comprising the following steps:

[0005] S1: Increase the contrast between the polar body and the background cytoplasm and cell membrane by subtracting the positive and negative defocus images to obtain a positive and negative defocus difference image. After binarization, the polar body is located based on the polar coordinate information of the detected polar body and background contour in the binarized image.

[0006] S2: Perform force analysis on the polar body to determine the direction of motion required for the microneedle to approach the polar body within the cell, and determine the trajectory of the microneedle approaching the polar body through finite element simulation modeling.

[0007] S3: The microneedle pierces the cell, and the motion control algorithm of the self-disturbance controller is used to compensate for the disturbance and error during the movement of the microneedle, correct the movement trajectory of the microneedle, and make the microneedle approach the polar body along the movement trajectory determined by S2; the microneedle extracts the polar body.

[0008] Furthermore, in step S1, under the microscope, the cells are moved to make the polar body appear in the focal position, the positive and negative defocus amounts are changed, and a positive defocus image and a negative defocus image are obtained. The difference between the two is calculated to obtain a positive and negative defocus difference image, which is then binarized to obtain a binarized image.

[0009] Furthermore, by comparing the errors between the polar body center positions obtained under different defocus amounts and the dyed polar body center positions obtained under focused conditions, the defocus amount corresponding to the minimum error is taken as the positive and negative defocus amounts.

[0010] Furthermore, in step S1, a polar coordinate system O is established. C -rθ, where O C Let r be the distance from the target point to the cytoplasmic center, θ be the angle between the line connecting the target point and the center and the -X axis. Contour segments of the polar body, cytoplasm, and zona pellucida are identified in the binarized image. Contour segments with excessively small areas are removed. The polar coordinates (r) of each point in the remaining contour segments are calculated. ij ,θ ij ), based on θ for each contour segment imin θ imax r imin and r imax The contour segments of the cytoplasm and the zona pellucida are determined, and the contour segment with the largest area located between the cytoplasm and the zona pellucida is taken as the contour segment of the polar body. Generally, contour segments with too small an area are less than 10 pixels.

[0011] Furthermore, the classification process for contour segments is as follows:

[0012] a. Determine the initial contour fragment of the cytoplasm: using θ imin Sort all contour segments, then from θ imin Search among the three smallest contour segments (r imin +r imax The smallest outline fragment is ) / 2, and it is used as the first outline fragment of the cytoplasm.

[0013] b. Determine the remaining contour segments of the cytoplasm: then follow the θ of the first contour segment. max Find the next cytoplasmic contour fragment in a clockwise direction, and assign θ to each contour fragment. min θ with the first contour segment max Take the difference; if the difference is minimized, and the (r) of the two contours is minimized... imin +r imax If the difference of ) / 2 is also less than the fixed threshold, then it is considered that the second contour segment of the cytoplasm in the clockwise direction has been found. Repeat this step until the remaining contour segments of all cytoplasm have been found.

[0014] If no other contour fragments of the next cytoplasm are found after traversing the remaining contour fragments, return to step a and select θ. imin Search among the three smallest contour segments (r imin +r imax ) / 2 The second smallest outline fragment, as the first outline fragment of the cytoplasm, is used to repeat step b;

[0015] c. Determine the outline segment of the transparent band: Arrange the remaining outline segments according to θ min Sort again from smallest to largest, starting from θ min Finding the three smallest contour segments (r imin +r imax The maximum value of ) / 2 is used as the first contour segment of the transparent band outline. The same method as step b is repeated to find the remaining contour segments of the transparent band.

[0016] d. Determine the outline segments of the polar bodies: Sort the outline segments outside the cytoplasm and zona pellucida in descending order of area, starting with the largest segment. Based on the θ of the outline segment... min θ max and (r) imin +r imax The value of ) / 2 determines the outline segment with the largest area located between the cytoplasm and the zona pellucida as the outline segment of the polar body.

[0017] Furthermore, in step S2, the cells undergoing force analysis are fixed by the suction needles, the cytoplasm and cell membrane are considered as elastic bodies, and the microneedles are simplified as rigid bodies.

[0018] Furthermore, in step S2, the center O of the microneedle opening is taken. P Establish the microneedle coordinate system O P -X P Y P , where Y P X represents the direction of microneedle movement. P Perpendicular to Y P ,

[0019] When the microneedle moves towards the polar body direction within the cell d P1 At that time, the displacement d of the polar body relative to the microneedle R

[0020]

[0021] Among them, k1 is related to the compression deformation of the cytoplasm, and k2 is related to the deformation of the zona pellucida.

[0022] The angle at which the polar body approaches the microneedle is determined by d. R With -Y P The included angle α is determined.

[0023] α=arctan(k1k2 / (1-k1))

[0024] The relative distance d between the polar body and the needle opening satisfies

[0025]

[0026] Where d0 is the initial distance between the center of the microneedle opening and the center of the polar body.

[0027] When the microneedle approaches the polar body at an angle β,

[0028] α=β-arctan(k1k2 / (1-k1))

[0029] By controlling β to make α zero, the polar body moves towards the center of the microneedle opening.

[0030] By finite element simulation of the motion trajectory of the polar body relative to the microneedle during the approach of the microneedle, k1 and k2 can be determined, and thus the direction of movement β of the microneedle can be determined.

[0031] Once d0, k1, and k2 are determined, the value of dp1 when d is 0 is determined based on β determined by finite element simulation, and then the motion trajectory of the microneedle approaching the polar body is determined.

[0032] Furthermore, in step S3, the arm drive system with microneedles is modeled as a second-order system. The movement speed of the microneedles is adjusted by an active disturbance rejection controller to correct the trajectory of the microneedles. The position disturbance of the microneedles caused by the uncertainty of the arm drive system is estimated by an extended state observer and compensated to the arm drive system.

[0033] Furthermore, in step S3, the position of the microneedle tip is indirectly located by the liquid level position of the microtube, and the microneedle tip is automatically controlled to approach the electrode along the motion trajectory determined in S2, and then the electrode is extracted.

[0034] Compared with the prior art, the beneficial effects of this invention are:

[0035] 1. This invention allows manipulation of the polar bodies of cells under a microscope while they are fixed in a suction needle state. The microneedle can be moved to a suitable position for removing the polar bodies without staining them. This avoids the difficulties caused by photobleaching and fluorescence fields resulting from staining, which can affect the movement of the microneedle and the calculation of the amount of cytoplasm removed. It also reduces the damage to oocyte viability caused by excessive cytoplasm removal.

[0036] 2. The intracellular movement trajectories of the microneedles determined in this invention are all normalized to the cytoplasmic radius; therefore, this method is applicable to oocytes of different sizes. This invention can determine the corresponding intracellular movement trajectory of the microneedles offline based on the position of the polar body, without relying on the operator's experience. This invention requires simple experimental equipment; conventional microneedles can be used to manipulate the polar body without manual intervention and without affecting subsequent cell manipulation.

[0037] 3. Using the microneedle of this invention to approach and extract the polar body of a domestic pig oocyte, the success rate of extracting the intracellular polar body is doubled compared to manual operation, and the average amount of cytoplasm removed is only 60% of that of manual operation. Attached Figure Description

[0038] Figure 1 This is a flowchart of the present invention;

[0039] Figure 2 These are the defocusing and focusing imaging effect diagrams of the present invention;

[0040] Figure 3 This is a flowchart and result diagram of the polar body positioning based on defocus imaging of the present invention;

[0041] Figure 4 This is a schematic diagram of the force analysis of the polar body obtained when the microneedles of the present invention move within the cell;

[0042] Figure 5 This is a structural diagram of the ADRC controller of the present invention;

[0043] Figure 6 This is a diagram illustrating the precise extraction process of oocyte polar bodies according to the present invention. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Embodiments of the present invention provide a method for precise extraction of cell polar bodies, such as... Figure 1 As shown, it includes the following steps:

[0046] S0: Oocyte preparation.

[0047] The cells used in this embodiment were porcine oocytes obtained from a local slaughterhouse. The extraction method was as follows: After the porcine ovaries were removed from the slaughterhouse, they were transported to the laboratory within two hours in a thermos containing physiological saline at 35°C to 37°C. They were then immediately washed twice with sterile physiological saline at 37°C containing 100 IU / L penicillin and 50 mg / L streptomycin. Oocytes were extracted from follicles with a diameter of 2-6 mm from the ovaries. The extracted cells were washed three times with TL-Hepes-PVA and then cultured in vitro for maturation (IVM) at 39°C and 5% CO2 for 42 hours. After IVM, the cells were de-ovulated using 0.1% hyaluronidase. Finally, the cells were washed three times with M199, and 20 oocytes with polar bodies were selected for use.

[0048] Staining of 10 oocytes requiring manual manipulation: MII stage oocytes from each experimental group to be tested were transferred into TCM199 containing 5 μg / ml Hoechst33342 and incubated for 15 min to complete the staining of the nucleus and polar body.

[0049] S1: Before extracting the polar body, under the microscope, move the cells to bring the polar body into focus, such as... Figure 2 As shown, by changing the positive and negative defocus amounts, positive defocus images and negative defocus images are obtained. The difference between the two is used to obtain a positive and negative defocus difference image. The difference between the positive and negative defocus images increases the contrast between the polar body and the background cytoplasm and cell membrane.

[0050] The contrast ratio C(x,y) can be determined by the following formula:

[0051]

[0052] Where I(x,y) is the gray value of the defocused image at coordinates (x,y), and I0(x,y) is the gray value of the focused image at coordinates (x,y); Δf is the defocus amount; Δn is the difference in refractive index between the polariton and the background; ▽ 2 h(x,y) is the surface curvature of the polar body.

[0053] In formula (1), the polar body is approximated as an isotropic material, so Δn can be considered a constant value. Observations show that the polar body is approximately spherical, therefore the surface curvature ▽ 2 h(x,y) can also be considered a constant value. Therefore, according to formula (1), the contrast C(x,y) between the polar body and the background is proportional to the defocusing amount Δf.

[0054] When the image is in a positive defocus state, the positive defocus contrast C + (x, y) satisfy:

[0055]

[0056] When the image is in a negative defocus state, the negative defocus contrast C - (x, y) satisfy:

[0057]

[0058] To increase the contrast between the polari and the background, the positive and negative image differences between the defocused images are used; the increased contrast ΔC(x, y) is obtained:

[0059]

[0060] In this embodiment, before performing positive and negative defocusing, it is necessary to determine the value of the defocus amount Δf: too small a defocus amount Δf will not produce sufficient contrast, leading to positioning errors, while too large a defocus amount will cause the polar body outline to be too blurry, increasing the positioning error. Therefore, it is necessary to compare the error between the polar body center position obtained under different defocus amounts and the stained polar body center position obtained under focused conditions, and take the defocus amount corresponding to the minimum error as the positive and negative defocus amounts for defocus imaging. Once this value is determined, it will not be changed for different individual cells of the same type of cell, provided that the object being operated on remains unchanged.

[0061] The positive and negative defocus difference images are binarized to obtain a binarized image. The polar coordinates of the detected polar body and background contours in the binarized image are used to locate the polar body.

[0062] To facilitate the positioning of the polar body, such as Figure 3 As shown, establish the image coordinate system O-XY and the polar coordinate system O C -rθ, where O C Let r be the distance from the target point to the cytoplasmic center, and θ be the angle between the line connecting the target point and the center and the -X axis. Contour segments of the polar body, cytoplasm, and zona pellucida are identified in the binarized image. Contour segments with excessively small areas are removed. For the coordinates (x, y) of points in the remaining contour segments... ij ,y ij ), calculate its polar coordinates (r) according to the following formula. ij ,θ ij ).

[0063]

[0064]

[0065] Where i is the number of the contour segment, j is the number of the point inside the contour segment, and (x0, y0) is the cytoplasmic center.

[0066] The polar coordinates (r) of each point ij ,θ ij ), determine θ for each contour segment imin θimax r imin and r imax Then, the contour segments are classified to determine the contour segments of the cytoplasm and the zona pellucida. Finally, the contour segment with the largest area located between the cytoplasm and the zona pellucida is taken as the contour segment of the polar body.

[0067] The classification process for contour segments is as follows:

[0068] a. Determine the initial contour fragment of the cytoplasm: using θ imin Sort all contour segments, then from θ imin Search among the three smallest contour segments (r imin +r imax The smallest outline fragment is ) / 2, and it is used as the first outline fragment of the cytoplasm.

[0069] b. Determine the remaining contour segments of the cytoplasm: then follow the θ of the first contour segment. max Find the next cytoplasmic contour fragment in a clockwise direction, and assign θ to each contour fragment. min θ with the first contour segment max Take the difference; if the difference is minimized, and the (r) of the two contours is minimized... imin +r imax If the difference of ) / 2 is also less than the fixed threshold, then it is considered that the second contour segment of the cytoplasm in the clockwise direction has been found. Repeat this step until the remaining contour segments of all cytoplasm have been found.

[0070] If no other contour fragments of the next cytoplasm are found after traversing the remaining contour fragments, return to step a and select θ. imin Search among the three smallest contour segments (r imin +r imax ) / 2 The second smallest outline fragment, as the first outline fragment of the cytoplasm, is used to repeat step b;

[0071] c. Determine the outline segment of the transparent band: Arrange the remaining outline segments according to θ min Sort again from smallest to largest, starting from θ min Finding the three smallest contour segments (r imin +r imax The maximum value of ) / 2 is used as the first contour segment of the transparent band outline. The same method as step b is repeated to find the remaining contour segments of the transparent band.

[0072] d. Determine the outline segments of the polar bodies: Sort the outline segments outside the cytoplasm and zona pellucida in descending order of area, starting with the largest segment. Based on the θ of the outline segment... min θ max and (r) imin +rimax The value of ) / 2 determines the outline segment with the largest area located between the cytoplasm and the zona pellucida as the outline segment of the polar body.

[0073] S2: Perform force analysis on the polar body to determine the direction of motion required for the microneedle to approach the polar body within the cell. Use finite element simulation modeling to determine the trajectory of the microneedle as it approaches the polar body. The cell undergoing force analysis is fixed by the holding needle; the cytoplasm and cell membrane are considered elastic bodies, and the microneedle is simplified as a rigid body.

[0074] Take the center of the microneedle opening O P Establish the microneedle coordinate system O P -X P Y P , where Y P X represents the direction of microneedle movement. P Perpendicular to Y P ,like Figure 4 As shown, when the microneedle moves towards the polar body within the cell, d P1 At that time, the polar body, in addition to the displacement d caused by the thrust along the direction of microneedle movement, P2 In addition, the displacement d caused by the compression of the zona pellucida due to cell deformation caused by the movement of microneedles S Since the main deformation of the zona pellucida leads to cell elongation due to microneedle movement, and the direction of cell elongation is basically consistent with the direction of microneedle movement, the polar body displacement d generated by the zona pellucida compression force... S The direction is considered to be perpendicular to Y. P Assume the distance the microneedle travels is d. P1 , then d P2 =k1d P1 d S =k1k2d P1 Among them, k1 is related to the compression deformation of the cytoplasm; k2 is related to the deformation of the zona pellucida.

[0075] From geometric relationships, the displacement d of the polar body relative to the microneedle can be determined. R

[0076]

[0077] The angle at which the polar body approaches the microneedle is determined by d. R With -Y P The included angle α is determined and can be calculated using the following formula.

[0078]

[0079] According to the law of cosines, the relative distance d between the polar body and the needle opening satisfies...

[0080]

[0081]

[0082] d0 is the initial distance between the center of the microneedle opening and the center of the electrode. Therefore, it can be seen that when the microneedle moves directly towards the electrode, due to the compression of the zona pellucida, the electrode deviates from the microneedle at an angle α. When the microneedle approaches the electrode at an angle β, the angle α between the electrode and the microneedle can be calculated using the following formula.

[0083] α=β-arctan(k1k2 / (1-k1)) (7)

[0084] Therefore, by controlling β to make α zero, the electrode moves towards the center of the microneedle opening. By determining k1 and k2 through finite element simulation of the electrode's trajectory relative to the microneedle as the microneedle approaches, the direction of movement β of the microneedle can be determined. Similarly, the relative distance d between the electrode and the microtube can be calculated using the following formula.

[0085]

[0086] Once d0, k1, and k2 are determined, the value of dp1 when d is 0 is determined by formulas (4) and (8) based on β determined by finite element simulation, which is the movement distance required to control the microneedle to reach the position of the electrode.

[0087] S3: The microneedle pierces the cell, and the motion control algorithm of the self-disturbance controller is used to compensate for the disturbance and error during the movement of the microneedle, correct the movement trajectory of the microneedle, and make the microneedle approach the polar body along the movement trajectory determined by S2; the microneedle extracts the polar body.

[0088] Considering that the arm drive system (micromanipulation arm stepper motor and lead screw system) with microneedles has second-order system characteristics, this embodiment models it as a second-order system and expands the ADRC design to use an active disturbance rejection controller to adjust the movement speed of the microneedles to correct their trajectory. Figure 5 As shown.

[0089]

[0090] Where x1 is the microneedle position, x2 is the microneedle speed, u is the stepper motor input step value, and f is the system disturbance.

[0091] Let ξ1=x, ξ3=f,ξ=[ξ1 ξ2 ξ3] T If f is differentiable, then the system space can be defined as follows:

[0092]

[0093] in, C = [1 0 0], The system's extended state observer (ES0) is

[0094]

[0095] Where L = [l1 l2 l3] T Let be the gain matrix of the observer. The gain should be chosen so that the characteristic polynomial satisfies the Hurwitz criterion, i.e.

[0096] λ0(s)=s 3 +l1s 2 +l2s+l3=(s+00) 3 (12)

[0097] Here, ω0 is the bandwidth of the system observer, and L takes the value of

[0098] L=[3ω0 3ω0 3 ω0 3 ] T (13)

[0099] When the observer's output can track ξ1 and ξ2 respectively, ξ3 can be accurately estimated. The object can then be simplified to a unity-gain integrator that makes the estimation error zero.

[0100]

[0101] at this time Therefore, the control law of ADRC is determined as follows:

[0102]

[0103] r represents the trajectory for setting the sealing resistance value, and K1 and K2 are respectively for making s 2 The root of +K2s+K1 is the controller gain that satisfies the Herwitz criterion. K1=ω c 2 K2=2ω c ω c This refers to the controller bandwidth.

[0104] The process of precise extraction of oocyte polar bodies is as follows Figure 6 As shown, after the oocyte is held by the aspirator needle, the microneedle rotates the cell until the polar body appears at the 1-2 o'clock position. After the microneedle enters the cell from the 3 o'clock position, the intracellular position is fed back in real time using the liquid level and the relative position of the microneedle tip. The ADRC algorithm is used to control the microneedle to approach the target polar body along a set trajectory within the cell. Upon reaching the polar body, the system automatically controls the microneedle to extract the polar body and withdraw from the cell. Simultaneously, the system uses image processing to obtain the volume of extracted cytoplasm. If 50% of the cytoplasm is removed and the polar body is still not drawn into the needle, the polar body extraction is considered a failure.

[0105] In the experiment, 20 cells were manipulated, 10 using the trajectory defined in this invention and 10 using a manually determined trajectory. Using the trajectory defined in this invention, 8 polar bodies were successfully extracted, a success rate of 80%, while the manual trajectory resulted in only 4 successful extractions, a success rate of only 40%. Statistical analysis of the successful cases revealed that the average cytoplasm removal rate using the method of this invention was 12%, while the average removal rate using manual methods was 20%.

[0106] The present invention has been described in detail above through embodiments, but the content described is only an exemplary embodiment of the present invention and should not be considered as limiting the scope of the present invention. The scope of protection of the present invention is defined by the claims. Any technical solutions designed by those skilled in the art using the technical solutions described in the present invention, or designed by those skilled in the art under the inspiration of the technical solutions of the present invention, within the substance and protection scope of the present invention, to achieve the above-mentioned technical effects, or any equivalent changes and improvements made to the scope of the application, should still fall within the patent protection scope of the present invention.

Claims

1. A method for precise extraction of cell polar bodies, characterized in that, Includes the following steps: S1: Increase the contrast between the polar body and the background cytoplasm and cell membrane by subtracting the positive and negative defocus images to obtain a positive and negative defocus difference image. After binarization, the polar body is located based on the polar coordinate information of the detected polar body and background contour in the binarized image. S2: Perform force analysis on the polar body to determine the direction of motion required for the microneedle to approach the polar body within the cell, and determine the trajectory of the microneedle approaching the polar body through finite element simulation modeling. S3: The microneedle penetrates the cell, and the motion control algorithm of the active disturbance rejection controller is used to compensate for the disturbance and error during the movement of the microneedle, correct the movement trajectory of the microneedle, and make the microneedle approach the polar body along the movement trajectory determined by S2; the microneedle is then removed from the polar body. In step S1, a polar coordinate system is established. O C - rθ ,in, O C The cytoplasm center, r The distance from the target point to the center of the cytoplasm. θ It is the angle formed by the line connecting the target point to the center point and the -X axis. Contour segments of the polar body, cytoplasm, and zona pellucida are identified in the binarized image. Contour segments with an area less than 10 pixels are removed, and the polar coordinates of each point in the remaining contour segments are calculated. r ij , θ ij ), based on each contour segment θ imin , θ imax , r imin and r imax Determine the outline segments of the cytoplasm and the zona pellucida, and take the outline segment with the largest area located between the cytoplasm and the zona pellucida as the outline segment of the polar body. The classification process for contour segments is as follows: a. Determine the initial outline of the cytoplasm: using θ imin Sort all contour fragments, then from θ imin Search among the three smallest contour segments ( r imin + r imax The smallest outline fragment is () / 2, and it is used as the first outline fragment of the cytoplasm. b. Identify the remaining contour segments of the cytoplasm: then follow the sequence of the first contour segment. θ max Find the next outline fragment of the cytoplasm in a clockwise direction, and then... θ min With the first outline fragment θ max Take the difference; if the difference is minimized, and the two contours are ( r imin + r imax If the difference between 1 / 2 is also less than the fixed threshold, then it is considered that the second contour segment of the cytoplasm in the clockwise direction has been found. Repeat this step until all the remaining contour segments of the cytoplasm have been found. If no further contour fragments of the next cytoplasm are found after traversing the remaining contour fragments, return to step a, starting from... θ imin Search among the three smallest contour segments ( r imin + r imax ) / 2 The second smallest outline fragment, as the first outline fragment of the cytoplasm, is used to repeat step b; c. Determine the outline segment of the transparent band: Arrange the remaining outline segments according to... θ min Sort again from smallest to largest, starting from... θ min Search among the three smallest contour segments ( r imin + r imax The maximum value of ) / 2 is used as the first outline segment of the transparent band, and then the first transparent band outline segment is used as the maximum value of ) / 2. θ max Find the next outline segment of the transparent band in a clockwise direction, and then... θ min With the first outline fragment θ max Take the difference; if the difference is minimized, and the two contours are ( r imin + r imax If the difference between 1 / 2 is also less than the fixed threshold, then it is considered that the next transparent band contour segment in the clockwise direction has been found. Repeat this step until the remaining transparent band contour segments are found. d. Identify the outline segments of polar bodies: Sort the outline segments outside the cytoplasm and zona pellucida in descending order of area, starting with the largest segment. Based on the outline segments... θ min , θ max and( r imin + r imax The value of ) / 2 is used to determine the contour segment with the largest area located between the cytoplasm and the zona pellucida as the contour segment of the polar body.

2. The method for precise extraction of cell polar bodies as described in claim 1, characterized in that, In step S1, under the microscope, the cells are moved to make the polar body appear in the focal position, the positive and negative defocus amounts are changed, and positive defocus images and negative defocus images are obtained. The difference between the two is used to obtain a positive and negative defocus difference image, which is then binarized to obtain a binarized image.

3. The method for precise extraction of cell polar bodies as described in claim 2, characterized in that, By comparing the error between the polar body center position obtained under different defocus amounts and the dyed polar body center position obtained under focused conditions, the defocus amount corresponding to the minimum error is taken as the positive and negative defocus amounts.

4. The method for precise extraction of cell polar bodies as described in claim 1, characterized in that, In step S2, the cells undergoing force analysis are fixed by the suction needles, the cytoplasm and cell membrane are considered as elastic bodies, and the microneedles are simplified as rigid bodies.

5. The method for precise extraction of cell polar bodies as described in claim 1, characterized in that, In step S2, the center of the microneedle opening is taken. O P Establish a microneedle coordinate system O P - X P Y P ,in Y P The direction of microneedle movement. X P Perpendicular to Y P , When the microneedle moves towards the polar body within the cell d P1 At that time, the displacement of the polar body relative to the microneedle d R satisfy ； in, k 1. It is related to the compression deformation of the cytoplasm. k 2. Related to the deformation of the zona pellucida. The angle at which the polar body approaches the microneedle is determined by d R and- Y P The included angle α Sure, ； The relative distance between the polar body and the needle tip d satisfy ； in, d 0 represents the initial distance between the center of the microneedle opening and the center of the polar body. And when the microneedle is at an angle β When approaching the polar body, ； By controlling β make α The value is zero, causing the electrode to move towards the center of the microneedle opening. The motion trajectory of the polar body relative to the microneedle during the process of the microneedle approaching the polar body was determined by finite element simulation. k 1. k 2. This allows you to determine the direction of movement of the microneedle. β， when d After 0, k1 and k2 are determined, the value of dp1 with d=0 is determined based on β determined by finite element simulation, and then the motion trajectory of the microneedle approaching the polar body is determined.

6. The method for precise extraction of cell polar bodies as described in claim 1, characterized in that, In step S3, the arm drive system with microneedles is modeled as a second-order system. The movement speed of the microneedles is adjusted by an active disturbance rejection controller to correct the trajectory of the microneedles. The position disturbance of the microneedles caused by the uncertainty of the arm drive system is estimated by an extended state observer and compensated to the arm drive system.

7. The method for precise extraction of cell polar bodies as described in claim 1, characterized in that, In step S3, the position of the microneedle tip is indirectly located by the liquid level position of the microtube, and the microneedle tip is automatically controlled to approach the electrode along the movement trajectory determined in S2, and then the electrode is extracted.

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