Method for precise cell manipulation based on torque balance

Abstract

The present invention relates to the technical field of cell-level microscopic operations. Provided is a method for precise cell manipulation based on torque balance, the method comprising the following steps: S1, analyzing a torque balance condition at a critical cell holding state, and determining the minimum holding force required to keep a cell fixed without detaching from a holding pipette; S2, performing a stress analysis on the cell that is being manipulated, and determining the expression for the minimum manipulation force required to balance the torque acting on the cell; S3, determining the friction coefficients between the holding pipette and the cell, and between a manipulation needle and the cell; and S4, carrying out manipulation on the basis of torque balance so as to reduce damage to the cell and to improve the manipulation precision. The cell is fixed by using the minimum holding force, and the minimum manipulation force is applied to a target cell by the force site control of the manipulation needle, such that the cell is in a torque-balanced state. The average cell deformation during the manipulation is reduced to be 15% or less of that observed during manual operations, and the error in cell manipulation is reduced to be within 1°.

Description

Cell precise poking method based on torque balance TECHNICAL FIELD

[0001] The present application belongs to the field of cell-level micromanipulation technology, and particularly relates to a cell precise poking method based on torque balance. BACKGROUND

[0002] Cell poking is a key step to realize cell orientation adjustment and then to perform specific cell structure site operation. In the cell poking operation, an operator usually uses a holding needle to fix one end of a cell and uses a poking needle to contact and poke the cell from the other end to make the cell rotate, and then adjusts the cell to the target orientation step by step. During the poking process, a suitable poking force needs to be applied to the cell to make the cell rotate while overcoming the friction force generated by the holding needle and to be in a torque balance state, otherwise the cell is easy to fall off from the holding needle and cause the poking failure. Meanwhile, in order to reduce the mechanical damage to the cell during the poking process, the poking force applied to the cell should be as small as possible under the premise of keeping the cell in the torque balance state.

[0003] The current manual operator often designs the contact position and motion trajectory of the poking needle and the cell according to experience, which is easy to cause the cell to fall off or fail to rotate due to torque imbalance and thus cause the poking failure. We have tried to poke the cell by applying a sufficient poking force in the early stage, but since the poking force cannot be detected in real time and the specific friction coefficient of the injection needle and the holding needle cannot be measured online for each cell, it is impossible to design a personalized poking trajectory for the target cell.

[0004] In addition, the poking force is not controlled during the poking process, so the poking force applied to the cell is still significantly greater than the minimum poking force required for the cell poking, which is easy to cause a large cell deformation. Therefore, it is of great significance to develop a poking method to measure the friction coefficient of the cell and the two needles simply, to control the force and position of the poking needle during the poking process, to apply the minimum poking force to the cell, and to keep the cell in the torque balance state, so as to improve the poking precision and reduce the mechanical damage to the cell during the poking process.

[0005] SUMMARY

[0006] The present application is aimed at the problems of large poking error and mechanical damage to the cell caused by torque imbalance in the current cell poking operation, sets the holding force based on torque balance, controls the force and position of the poking needle, provides the minimum poking force required for the cell poking, keeps the cell in the torque balance state, and finally realizes a cell precise poking method based on torque balance, which reduces the cell poking error and the mechanical damage to the cell.

[0007] The present application adopts the following technical solution to solve the above problems:

[0008] A cell precise poking method based on torque balance, the method comprising the following steps:

[0009] S1: determining the minimum holding force required for fixing the cell based on torque balance condition: by analyzing the torque balance condition of the critical holding state of the cell, the minimum holding force required for fixing the cell so that it does not fall off the holding needle is determined;

[0010] The critical holding state is a state in which the holding pressure is reduced to just maintain the cell from falling off the tube opening of the holding needle, the cell deformation due to holding is ignored in the process of stress analysis, the holding needle is placed horizontally and the tube opening is completely covered by the cell surface, and there is no air leakage phenomenon. At this time, the liquid in the environment is regarded as a static fluid, and there is no additional fluid force on the cell. At this time, the forces acting on the cell only exist gravity, buoyancy and the holding force of the microtube. When the cell is in the critical holding state, the torque of the combined force of gravity and buoyancy that makes the cell fall off the holding needle is balanced with the fixed torque generated by the holding force, and the minimum holding pressure required for fixing the cell is calculated.

[0011] S2: determining the minimum poking force expression for balancing the cell torque: by analyzing the stress of the cell in the poking process, the minimum poking force expression required for balancing the cell torque is determined;

[0012] It is assumed that the friction coefficients of different positions on the cell surface with the holding needle and the injection needle do not change, the holding and contact deformation generated by the holding needle and the injection needle and the cell have reached a stable state, and the poking process is slow enough to allow the forces of the holding needle and the injection needle on the cell to be fully released to the cell. Therefore, in the stress analysis of the cell, the related theorem of statics is used to decompose the poking force into a normal pressure pointing to the center of the cell and a pushing force along the tangent direction. According to the translation theorem of statics, the poking force can be translated to the center of the cell and further decomposed into two forces in X and Y directions and a rotational torque, wherein the two forces are balanced with the holding force of the holding needle and the friction resistance between the holding needle and the cell, respectively. According to the torque balance condition of the friction resistance torque and the friction pushing torque, the expression of the minimum friction pushing force and the normal pressure required for poking the cell is finally determined, that is, the minimum poking force.

[0013] S3: determining the friction coefficients between the holding needle-cell and the poking needle-cell through friction pushing experiment: by performing a friction pushing experiment on the target cell, the friction coefficients between the holding needle-cell and the poking needle-cell in the minimum poking force expression in S2 are determined;

[0014] In the friction experiment, the normal extrusion force is exerted on the fixed target cell by the poking needle, and then the tangential movement is measured, the resultant force of the friction force and the extrusion force is calculated, and the friction coefficient between the poking needle and the cell is determined by the ratio of the friction force to the extrusion force. In the pushing experiment, the normal extrusion force is gradually increased by the poking needle, and then the cell is pushed in the tangential direction, and when the cell starts to rotate, the sliding friction force of the holding needle is calculated by using the balance condition of the pushing moment and the friction moment. The friction coefficient between the holding needle and the target cell is determined by the ratio of the sliding friction force to the pressure on the holding needle.

[0015] S4: Poking based on moment balance, reducing the damage to the cell and improving the poking precision: In the poking process, the cell is fixed using the minimum holding force, the force position control of the poking microneedle is performed according to the detected poking force and the contact point position, and the cell is always in the state of moment balance, so that the minimum poking force is exerted on the cell, the mechanical damage to the cell is reduced, and the poking precision is improved.

[0016] Before the poking starts, the minimum holding force required for fixing the cell is calculated according to the cell density and the measured cell geometric parameters, and then the minimum poking force is determined according to the minimum holding force, the friction coefficient and the contact point position. The minimum poking force required by the cell is exerted on the cell by the force position control of the poking needle, and the cell is separated from the contact after the cell is rotated by a certain angle. In the poking process, the pushing force is detected by the micro force sensor, the depth of the poking needle is adjusted, the minimum poking force is always exerted on the cell to keep the cell moment balance, the cell is rotated smoothly, the actual cell rotation angle is calculated by the angular displacement of the cell edge feature in the focusing state, and the cell deformation amount in the poking process is estimated according to the depth of the microneedle pressed into the cell surface.

[0017] The beneficial effects of the present application are:

[0018] 1. In the present application, the holding force is set based on the moment balance, the minimum holding force is used to fix the cell, the force position control of the poking needle is performed, the minimum poking force is exerted on the target cell by the force position control of the poking needle, the cell is in the balanced state, the average cell deformation in the poking process is reduced to less than 15% of the manual operation, and the cell poking error is reduced to within 1°.

[0019] 2. In the present application, the friction coefficient between the surface of each target cell and the holding needle and the poking needle can be measured online through a simple friction pushing experiment, and then the personalized minimum poking force required for balancing the moment of each target cell in the poking process is obtained, so that the poking precision is ensured and the mechanical damage to the cell in the poking process is reduced.

[0020] 3、The holding force applied to the cell by the application is the minimum holding force required to fix the cell, which reduces the holding damage to the cell and greatly reduces the poking force required to poke the cell, thereby reducing the mechanical damage to the cell during the poking process.

[0021] 4、The force position of the poking needle is controlled according to different contact points and feedback poking force values during the poking process, so that the minimum poking force can be applied to the cell, the control error of the poking force is reduced, and the accuracy of the poking is ensured.

[0022] 5、The total cost of the self-made holding needle, poking needle and micro force sensor in the application is only more than 300 yuan, the operation tool cost is low, and the application of the cell poking method is conducive to the popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present application, the drawings needed in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Fig. 1 is a flow chart of cell precise poking based on torque balance;

[0025] Fig. 2 is a schematic diagram of torque balance in the critical holding state of spherical cells;

[0026] Fig. 3 is a schematic diagram of cell stress analysis during the poking process;

[0027] Fig. 4 is a schematic diagram of friction pushing experiment of the application;

[0028] Fig. 5 is a control block diagram of cell precise poking based on torque balance;

[0029] Fig. 6 is an experimental diagram of cell precise poking based on torque balance;

[0030] Fig. 7 is a cell deformation variation diagram during the poking process. DETAILED DESCRIPTION

[0031] In order to make those skilled in the art better understand the technical solutions of the present application, the present application will be described in detail below in combination with the drawings and specific embodiments.

[0032] As shown in Fig. 1, the present application discloses a cell precise poking method based on torque balance, which comprises the following steps:

[0033] S1: determining the minimum holding force required to fix the cell based on torque balance condition: by analyzing the torque balance condition of the critical holding state of the cell, the minimum holding force required to fix the cell so that it does not separate from the holding needle is determined;

[0034] S2: Determine the minimum plucking force expression required to balance the cell torque: By analyzing the force on the cell during the plucking process, determine the minimum plucking force expression required to balance the cell torque;

[0035] S3: Friction pushing experiment to determine the friction coefficient between the holding needle and the cell and the moving needle and the cell: By performing a friction pushing experiment on the target cell, the friction coefficient between the holding needle and the cell and the moving needle and the cell in the minimum moving force expression in S2 is determined;

[0036] S4: Plugging based on torque balance reduces damage to cells and improves plucking accuracy: Use minimum suction force to fix the cells during the plucking process, and control the force position of the plucking microneedle according to the detected plucking force and contact point position to keep the cells in a torque balance state at all times, thereby ensuring that the minimum plucking force is applied to the cells, reducing mechanical damage to the cells while improving the plucking accuracy.

[0037] The operation steps of this embodiment are as follows:

[0038] S0: Preparation of target cells.

[0039] The target cells used in this embodiment are porcine oocytes, which were obtained from a local slaughterhouse. After the porcine ovaries were removed from the slaughterhouse, they were transported to the laboratory within two hours in a thermos containing 35° to 37° physiological saline. They were then immediately washed twice with 37° sterile physiological saline containing 100 IU / L of penicillin and 50 mg / L of streptomycin. Oocytes were extracted from ovarian follicles with a diameter of 2-6 mm. The extracted cells were rinsed three times with TL-Hepes-PVA and then subjected to in vitro maturation (IVM) for 42 hours in an incubator at a temperature of 39° and a carbon dioxide concentration of 5%. After IVM, the cells were de-oviposited with 0.1% hyaluronidase. Finally, the cells were washed three times with M199, and oocytes with polar bodies were selected as target cells for the experiment.

[0040] S1: Obtaining the minimum holding force based on the torque balance condition of the critical holding state.

[0041] As shown in Figure 2, a horizontally placed suction needle is used to apply a suction force F to the target oocyte. H Fixed cells, because the density of cells is greater than the density of culture medium, the gravity G of cells and buoyancy F F The direction of the resultant force will be parallel to the Z axis and downward, which will cause the deformation of the cell body in contact with the lower half of the inner wall of the suction needle (the part close to H2) to be greater than that of the upper half (the part close to H1). N2 The force f applied by the upper part will be greater than N1 , at this time the elastic force FN The direction of the resultant force will be biased toward the positive direction of the Z axis, and its point of action will be biased toward the side where H2 is located.

[0042] When the cell force reaches equilibrium and the shape no longer changes, according to the elastic body rigidification theory, the cell at this time can be equivalent to a rigid body. From the force balance formula of the rigid body, we can know that

[0043] Since the combined force of gravity and buoyancy is parallel to the Z axis, F H Parallel to the X axis and in the XZ plane, according to the three-force equilibrium theorem of rigid body, we know that F N Also in the XZ plane and its line of action passes through G and F F and F H The intersection of the lines of action. Its size satisfies the following relationship

[0044] At this time, since the cell does not rotate, the oocyte not only satisfies the force balance, but also the moment balance. F The net force is F GF Taking the lowest point H2 between the cell and the inner wall of the holding needle as the moment center, then F H 、F GF and F N The torque m generated about H2 H2 (F H ), m H2 (F GF ) and m H2 (F N ) must be balanced, that is, there is m H2 (F H )+m H2 (F GF )+m H2 (F N )=0 (3)

[0045] Among them F GF For G and F F The resultant force of . That is, F H *R H_in =(GF F )*L+F N *L N (4)

[0046] When the holding force F H When it starts to decrease, the elastic force F N The holding force F will also decrease, and its function will gradually change from preventing cells from entering the holding needle to fixing the cells so that they do not fall off. H After decreasing to a certain extent, starting from point H1, the F applied by the inner wall of the holding needle to the cellN Close to zero. As F H The force of the holding needle edge near H2 continues to decrease, and finally only the elastic force generated by the holding needle edge near H2 is greater than zero, so at this time F N The action point can be approximately considered to be H2, F H The moment m about point H2 H2 (F H ) approaches zero. As shown in Figure 2, when the cell is just not separated from the holding needle, the cell reaches the critical holding state, at which time F H , G and F F Regarding the moment balance of H2, m H2 (F H )+m H2 (F GF )=0 (5)

[0047] That is, F H *R H_in =(GF F )*L (6)

[0048] If F H If the force continues to decrease, the cell will first detach from H1, then gradually detach from the other parts of the holding needle, and finally detach from H2, and finally slide out of the holding needle. The minimum holding force F required to hold the cell without falling off can be calculated from formula (6): Min_H , the expression is as follows

[0049] The experiment found that when the cell is in the critical holding state, the deformation of the cell caused by the holding needle is small, and the cell can be approximately spherical. According to the geometric relationship,

[0050] S2: Obtaining the minimum shifting force based on torque balance.

[0051] As shown in Figure 3, the force F acting on the cell I Can be divided into two parts: normal extrusion force F In and tangential thrust F Iτ , where the normal extrusion force F In Pointing to the center O of the cell, the angle between its line of action and the X axis is θ1, F I The two components satisfy the following relationship

[0052] Let F I and F In The angle between them is α, then according to the geometric relationship, α=arctan(F Iτ / F In ) (10)

[0053] According to the parallel force line theorem, F I It can be equivalent to the force F with the same magnitude and direction as the point of action at the center of gravity O. I ′ and the clockwise couple m on O o (F I ), where m o (F I ) is of a size that satisfies m o (F I )=F I *D I (11)

[0054] Among them D I F I The force arm of O, when only considering the penetration deformation, is shown in Figure 3. It is not difficult to obtain D from the geometric relationship. I =(R0-D In )sinα (12)

[0055] Substituting formula (12) into formula (11) we can get m o (F I )=F I *D I =F Iτ *(R0-D In ) (13)

[0056] Among them F I ' Component force F' along the X axis I1 and the force component F′ along the Z axis I2 The size satisfies the following formula F′ I1 =F I ′*cos(θ1-α)=F In *cos(θ1-α) / cosα (14)

[0057] F′ I2 =F I ′*sin(θ1-α)=F In *sin(θ1-α) / cosα (15)

[0058] Similarly, the force applied by the holding needle is equivalent to two mutually perpendicular forces whose action points are at the intersection of the X-axis and the holding needle port plane: the elastic force F parallel to the X-axis N and the friction force F parallel to the Z axis S and a couple m that can rotate the cell clockwise around the center O o According to the force balance condition F′ I1 and F′ I2 F′ should be satisfiedI1 = F N - F H (16) F' I2 = F S + F F - G (17)

[0059] In formula (17), since F F is less than G, F S is positive, and the direction is along the positive direction of Y axis, which is consistent with the direction of the friction force resisting the counterclockwise rotation of the cell by the holding needle, so it is possible to keep the cell in a balanced state by counterclockwise poking of the cell by the poking needle.

[0060] When the cell is in a balanced state, according to the moment balance condition in statics, it can be known that the additional force couple at the center O of the cell should satisfy the following relationship m o = F I * D I - F S * L (18)

[0061] wherein L is the distance from the center of the cell to the mouth of the holding needle, and when the deformation of the cell is not obvious, the following formula can be used for estimation according to the geometric relationship

[0062] Formulas (16)-(18) establish the conditions required to be satisfied by the acting force for keeping the cell balanced when the cell rotates counterclockwise. Substituting formulas (9), (12), (14)-(17) into formula (18) can obtain

[0063] In fact, in order to balance the action of F I , the resultant force of the friction force F S generated by the holding needle and the newly added elastic force F N1 should be on the same straight line as F I and equal in size, at which time the moment balance condition and the force balance condition are automatically satisfied. In the above formula, m o is actually the additional force couple generated by the translation of F N1 at point H according to the force line translation theorem, that is, m o = F N1 * D H = F' I1 * D H = F In * D H * cos(θ1-α) / cosα (21)

[0064] Substituting formula (20) into formula (21) and rearranging can obtain

[0065] In summary, when the force is applied on the upper part of the cell, the cell needs to be pushed counterclockwise to rotate, and when the applied force meets the formula (16)-(18), the cell can reach a state of force and torque balance. The condition for the cell to just be able to rotate is when the rotating torque m I provided by the injection needle is equal to the maximum resistance provided by the suction needle m Hmax , that is, m I = m o (F In ) = m Hmax (23)

[0066] And m Hmax is the friction coefficient μ H between the suction needle and the cell, which satisfies the following relationship: m Hmax = μ H *F N *L+m o = μ H *(F H +F′ I1 )*L+m o (24)

[0067] Substituting formula (15) and formula (21) into formula (24) can obtain:

[0068] By formula (13), formula (23) and formula (25), the expression of F In is as follows

[0069] As shown in Figure 3, the tangential pushing force F Iτ is generated by the normal pressure F In . At the same time, F In is also generated by the cell deformation D In . If D In is gradually increased first, and then the microneedle is controlled to move along the normal direction, the maximum friction force F Max_Iτ generated by F In satisfies the following relationship F Max_Iτ = μ I *F In (27)

[0070] Where μ I is the friction coefficient between the injection needle and the oocyte.

[0071] Substituting formula (10) and formula (26) into formula (27) can obtain the minimum tangential pushing force expression:

[0072] Correspondingly, the normal pressure F In that can generate the tangential pushing force in formula (28) is expressed as follows:

[0073] S3: Cell and microneedle friction coefficient acquisition based on friction pushing experiment.

[0074] As shown in FIG. 4, the present application obtains the friction coefficient μ I between the pushing needle and the cell surface through the friction experiment. In First, the cell is subjected to a large enough suction force by the suction needle to ensure that the cell does not rotate in the friction experiment, and the normal force F I is applied to the cell by the pushing needle. Iτ According to formula (9), the tangential pushing force F I and the friction coefficient μ H between the pushing needle and the cell can be calculated by the following formula.

[0075] The present application obtains the friction coefficient μ H between the suction needle and the cell through the pushing experiment. H First, the target oocyte is subjected to suction by using the suction pressure P H The suction force F H can be obtained by the following formula. 2 H_in P H (32)

[0076] By gradually increasing F In and pushing the cell along the tangent direction of the cell surface until the cell rotates, the pushing moment m I and the friction resistance moment m H reach equilibrium.

[0077] The resistance moment is m H = (F H +F’ I1 )*L*μ H (34)

[0078] Substituting (14), formula (19) and formula (32) into formula (34) can obtain

[0079] When m I and m H reach equilibrium, it can be known from formula (33) and formula (35) that

[0080] The contact point position θ of the poking needle with the cell, the cell radius R0, and the indentation depth D in the above process In obtained by the image processing algorithm, F In and F I The magnitude is obtained by the micro-force sensor detection, and α is obtained by calculation using equation (10).

[0081] S4: Precise cell poking process based on torque balance.

[0082] S41: Before the poking starts, the minimum holding force required to fix the cell is calculated according to equation (8) based on the cell and liquid density reported in the literature, the cell radius obtained by image processing, and the inner diameter of the holding needle.

[0083] S42: The friction coefficients of the target cell with the holding needle and the poking needle are obtained based on the minimum holding force and the detection by the friction pushing experiment, and the minimum poking force at different contact point positions is further calculated using equations (28) and (29) based on the minimum holding force and the above friction coefficients.

[0084] S43: As shown in FIG. 5, the position of the needle tip of the poking needle obtained by real-time image processing is controlled to contact the cell surface at the initial point and press into the cell surface to a certain depth until the normal pressure feedback by the micro-force sensor reaches the calculated value of equation (29).

[0085] Then the poking needle moves in the tangential direction to push the cell to rotate, and the indentation depth of the microneedle is controlled according to the calculation result of equation (29) at each point until the pressure reaches the calculated value. By the above force position cooperative control, the minimum required poking force is applied to the cell, and the cell is detached from the contact after rotating a certain angle. During the poking process, the pushing force is detected by the micro-force sensor, the indentation depth of the poking needle is adjusted, and the minimum poking force is always applied to the cell to maintain the torque balance of the cell, ensuring the smooth rotation of the cell. The actual cell rotation angle is calculated by the angular displacement of the cell edge feature in the focusing state, and the cell deformation during the poking process is estimated according to the depth of the microneedle indentation into the cell surface.

[0086] As shown in FIG. 6, by performing the poking experiment on 5 cells, the poking angle is set to gradually increase from 5° to 40°, with an increase of 5° at each step, and the average poking angle of all cells is set and actual, as shown in Table 1:

[0087] Table 1: Cell Poking Experiment Results

[0088] As can be seen from Table 1, the average poking error of the cell is only 0.9°, which is much smaller than the average poking error of 8.3° reported in previous manual poking operations.

[0089] The depth of penetration D of the poking needle on the cell surface is obtained according to the position of the poking needle tip during the poking process In and the result is taken as an index of the cell deformation.

[0090] As shown in Fig. 7, according to the measurement result of one cell, the average deformation of the present application is only 4.2 μm, far less than the average cell deformation of 28.2 μm in manual poking operation, only 15% of the latter.

[0091] The above has been described in detail through examples, but the content is only the preferred embodiment of the present application, and cannot be considered as used to limit the implementation range of the present application. Any equivalent changes and improvements made according to the application range of the present application should still belong to the patent coverage range of the present application.

Claims

1. A method for precise cell manipulation based on torque balance, characterized by: The method comprises the following steps: S1: Determine the minimum holding force required to fix the cell based on the torque balance condition: By analyzing the torque balance condition of the cell's critical holding state, determine the minimum holding force required to fix the cell so that it does not fall off the holding needle; S2: Determine the minimum plucking force expression required to balance the cell torque: By analyzing the force on the cell during the plucking process, determine the minimum plucking force expression required to balance the cell torque; S3: Friction pushing experiment to determine the friction coefficient between the holding needle and the cell and the moving needle and the cell: By performing a friction pushing experiment on the target cell, the friction coefficient between the holding needle and the cell and the moving needle and the cell in the minimum moving force expression in S2 is determined; S4: The movement is performed based on torque balance to reduce damage to cells and improve the movement accuracy.

2. The method for precise cell manipulation based on torque balance according to claim 1, characterized in that: In S1, the critical holding state is the state where the holding pressure is reduced to a level that can just prevent the cells from falling off from the nozzle of the holding needle. The holding needle is placed horizontally and the nozzle is completely covered by the cell surface without air leakage. The liquid in the environment is regarded as a static fluid.

3. The method for precise cell manipulation based on torque balance according to claim 2, characterized in that: The cell deformation caused by adhesion is ignored in the process of critical adhesion state force analysis.

4. The method for precise cell manipulation based on torque balance according to claim 1, characterized in that: In S2, it is assumed that the friction coefficients between the holding needle and the injection needle at different positions on the cell surface remain unchanged, and the movement process is slow enough so that the forces exerted by the holding needle and the injection needle on the cell have sufficient time to be released to the cell, so that the cell deformation has reached stability.

5. The method for precise cell manipulation based on torque balance according to claim 1, characterized in that: In S3, in the friction pushing experiment, a normal squeezing force is applied to a firmly fixed target cell by a moving needle, and then the needle is moved tangentially along the cell surface. The resultant force of friction and pressure is measured by a micro-force sensor, and the friction force is calculated by the resultant force and the squeezing force. The friction coefficient between the moving needle and the cell is determined by the ratio of the friction force to the squeezing force.

6. The method for precise cell manipulation based on torque balance according to claim 5, characterized in that: In S3, in the friction pushing experiment, the normal extrusion force is gradually increased by moving the needle and then the cell is pushed along the tangential direction. When the cell begins to rotate, the equilibrium condition of the pushing torque and the friction torque is used to calculate the sliding friction force of the holding needle, and the friction coefficient between the holding needle and the cell is determined by the ratio of the sliding friction force and the pressure on the holding needle.

7. The method for precise cell manipulation based on torque balance according to claim 1, characterized in that: In S4, the minimum holding force is used to fix the cells during the plucking process, and the force position of the plucking microneedle is controlled according to the detected plucking force and contact point position, so that the cells are always in a torque balance state, thereby ensuring that the minimum plucking force is applied to the cells, reducing mechanical damage to the cells while improving the plucking accuracy.

8. The method for precise cell manipulation based on torque balance according to claim 7, characterized in that: In S4, the dialing process includes the following steps: S41: Calculate the minimum holding force required to fix the cells before the plucking begins; S42: Determine the minimum required pulling force based on the minimum holding force, the friction coefficient between the cell and the two microneedles, and the initial contact point; S43: Apply a minimum holding force to the cell and perform force-position coordinated control on the moving needle to apply the required minimum moving force to the cell, move the cell to rotate a predetermined angle, and then break contact with the cell.

9. The method for precise cell manipulation based on torque balance according to claim 8, characterized in that: In S41, the minimum holding force required to fix the cells is calculated based on the cell density and measured cell geometry.

10. The method for precise cell manipulation based on torque balance according to claim 8, characterized in that: During the plucking process, the pushing force is detected by a micro-force sensor, and the actual cell rotation angle is calculated by the angular displacement of the cell edge features in the focused state relative to the cell center. The cell deformation during the plucking process is estimated based on the depth of the microneedle pressed into the cell surface.

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