Automatic blind method patch clamp operation method

Abstract

The invention discloses an automatic blind method patch clamp operation method, and belongs to the technical field of cellular level micromanipulation, and the method comprises the following steps: firstly, designing an animal head plate double-layer fixing device, through a plate double-layer mechanical structure, improving the fixing stability of an animal head, and reducing the influence of the movement of the animal head on patch clamp operation; secondly, a skull global plane image is obtained through an image splicing technology, and accurate positioning of a craniotomy hole is completed based on the image; planning a motion path of the electrode entering the brain environment through the craniotomy hole based on coordinate transformation, and finishing track planning of the microtubule electrode according to coordinate change; after the electrode enters the cerebral cortex, blood vessel obstacle avoidance, target cell contact, high-resistance sealing and membrane rupture are completed based on the electrode resistance, finally whole-cell electrophysiological signal recording is completed, and blind patch clamp automatic operation based on electrode resistance feedback is achieved. The sealing success rate is improved by 5.8%, and the recording success rate is improved by 3.7%.

Description

Technical Field

[0001] The invention belongs to the technical field of cell-level micromanipulation, and particularly relates to an automated blind patch clamp operation method. Background Art

[0002] Blind patch clamp technology is widely used in brain science and neuroscience research because it does not require expensive in vivo imaging microscopes and can detect animal brain nerve cells. However, the current blind patch clamp operation still faces the following challenges: First, in order to prevent the animal's head movement from interfering with the patch clamp operation, it is necessary to design an animal head fixation device, which must not only effectively fix the animal's head but also leave enough operating space for subsequent patch clamp operations; second, in the current process of manually controlling the electrode to enter the brain, due to the small craniotomy hole, the operator needs to carefully control the movement of the electrode to avoid collision with the fixation device and the skull, so the needle insertion process is extremely slow, which greatly affects the operation speed of the blind patch clamp; third, after the microtube electrode enters the animal's brain, it is difficult to distinguish the blood vessels and target cells encountered due to the lack of visual feedback, resulting in a low success rate of cell signal detection. The above shortcomings greatly reduce the efficiency of blind patch clamp operation and limit its promotion and application in animal brain science.

[0003] In summary, developing an effective animal head fixation device to automate the rapid brain entry of microtube electrodes and automated blind patch clamp operations that can identify target cells and blood vessels and perform corresponding operations is of great significance to improving the success rate of patch clamp operations and promoting their application in animal brain science. Summary of the invention

[0004] The present invention aims at the shortcomings of existing blind patch clamps, such as the lack of an effective animal head fixing device, the inability of electrodes to quickly enter the brain, and the inability to identify cells and obstacles and perform operations. An automated animal blind patch clamp operation method is proposed. The double-layer fixing device of the animal head plate is designed and processed to suppress the influence of the animal head movement on the patch clamp operation, and then the craniotomy hole is accurately located based on the global plane image of the skull, and the path for the electrode to enter the brain is planned based on coordinate transformation. After entering the brain, obstacle avoidance and sealing, membrane rupture and signal recording of target cells are completed based on electrode resistance feedback, and finally the automated blind patch clamp operation is completed.

[0005] The present invention adopts the following technical solutions to solve the above problems:

[0006] An automated blind patch clamp operation method, the method comprising the following steps:

[0007] S1: Design and manufacture a double-layer fixing device for the animal head plate to reduce the influence of the animal head movement on the patch clamp operation, fix the animal head on the double-layer fixing device for the animal head plate, and install electrodes to prepare for subsequent operations.

[0008] Combining the experience and practical problems of the artificial experimental process, the fixing device was designed and optimized. The device is divided into two parts: a head plate assembly and a head stage assembly. The head stage assembly is mainly used to support the head plate assembly and is provided with a groove that matches the head plate assembly. The two are fixed by a carbon fiber plate combination.

[0009] S2: The global planar image of the skull is obtained through image stitching technology, and the precise positioning of the craniotomy hole is completed based on the image.

[0010] After the 4x microscope focuses on the skull, it moves to the lower left side of the skull and sets the current position as the starting point; the 4x microscope moves from the starting point in a fixed order and collects local images of the skull at each point; the local images are gradually spliced ​​in two steps to cover the repeated parts; distortion is not considered during the splicing process; a complete skull plane image is obtained to determine the location of the cranial foramen.

[0011] S3: Plan the movement path of the electrode through the craniotomy hole into the brain environment based on coordinate transformation.

[0012] Coordinate systems are established for the electrode tip, skull plane, and image plane, and the coordinate origin is set. The target point of electrode movement is determined in the image coordinate system, and the relative coordinates of the target point and the current electrode tip in the same coordinate system are calculated by coordinate transformation, and the electrode movement is controlled accordingly.

[0013] S4: Based on the electrode resistance, the blood vessel is avoided and the target cells are contacted, sealed with high resistance and the membrane is broken, and finally the whole-cell electrophysiological signal recording is completed.

[0014] Whether the electrode has entered the cerebral cortex is determined based on the electrode resistance value. Once inside, it descends at a uniform speed and keeps a slight positive pressure on the microtube to prevent blockage of the tube opening. When encountering an obstacle or a target cell, the electrode rising speed is used to determine whether it is a target cell or an obstacle. If it is a blood vessel, the obstacle is avoided; if it is a nerve cell, automatic high-resistance sealing, membrane rupture and signal recording operations are completed based on the feedback of the electrode resistance value.

[0015] Furthermore, in S1, the double-layer fixing device for the animal head platform includes a two-layer structure of a head plate assembly and a head platform assembly, the head plate assembly is used to be bonded and connected to the animal's skull, and the head platform assembly supports the head plate assembly and is fixed to the platform.

[0016] Furthermore, a hollow elliptical structure is designed inside the head plate assembly. The hollow elliptical structure is surrounded by small holes for penetrating dental cement to bond the skull. The dental cement can completely bond with the skull. The hollow part leaves space for patch clamp operation. Four extension beams are provided between the head plate assembly and the head stage assembly.

[0017] Furthermore, the head stage assembly is provided with a placement groove matching the extension beam, and the head stage assembly is fixedly connected to the head plate assembly by screws and an auxiliary fixed high-toughness carbon fiber plate for installing and supporting the head plate assembly, and an electrode cleaning groove is provided on each side of the upper surface of the head stage assembly, and the electrode cleaning groove is used for cleaning operations of the microtube electrode.

[0018] Furthermore, in S2, the image stitching technology is specifically to collect m×n skull microscopic field images by controlling the platform to move back and forth in a zigzag manner, and stitch them together to form a global plane image of the skull including the craniotomy hole.

[0019] Furthermore, in S2, after the images are stitched, the center and range of the craniotomy hole are obtained by automatic contour detection, and the threshold of the contour detection is determined by the empirical value of the size of the craniotomy hole.

[0020] Furthermore, in S3, when planning the movement path of the electrode through the craniotomy hole into the brain environment, coordinate systems are established for the electrode tip, skull plane, and image plane respectively, and the target position and the electrode tip are placed in the same coordinate system through coordinate transformation, so as to plan the movement path and control the movement of the electrode to the target position.

[0021] Furthermore, in S4, whether the electrode has entered the cerebral cortex is determined based on the electrode resistance value. After entering, it is descended at a uniform speed and the microtube is kept at a slight positive pressure to prevent blockage of the tube mouth. When encountering an obstacle or a target cell, the microtube electrode resistance will increase significantly. The rising speed is used to determine whether the front end encounters the target nerve cell or the blood vessel. If the rising speed of the resistance value is less than the threshold, it is considered that the blood vessel is encountered, and the electrode will first withdraw and then move laterally to avoid the blood vessel. If the rising speed of the electrode resistance value exceeds the threshold, it is considered that the target nerve cell is encountered, and the system automatically stops the microtube positive pressure, first applies a stable low negative pressure to complete the high-resistance sealing, and then applies a high negative pressure pulse to break the membrane, and finally completes the whole-cell electrophysiological signal recording.

[0022] Compared with the existing blind patch clamp technology, the present invention has the following beneficial effects:

[0023] 1. The present invention designs a special fixing structure to solve the problem of animal head fixation, which effectively increases the contact area with the skull and improves stability while reserving the electrode operation space to the greatest extent, which helps to improve the fixation effect.

[0024] 2. The image stitching cranial hole positioning and electrode movement trajectory planning adopted in the present invention realize the rapid positioning of the cranial hole and the precise guidance of the electrode, effectively reducing the probability of collision when the electrode moves toward the target, and at the same time, no human intervention is required, greatly improving the speed of microtubules entering the brain.

[0025] 3. The blind patch clamp automated operation process designed by the present invention realizes obstacle avoidance and target operation of the electrode in the brain tissue without relying on the operator's experience. Compared with manual operation, the blind patch clamp experiment using the present invention shortens the experimental time by 46% under the same conditions, increases the sealing success rate by 5.8%, and increases the recording success rate by 3.7%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the description of the specific embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is a flow chart of the present invention;

[0028] Figure 2 It is a structural schematic diagram of a head plate assembly of a double-layer fixed structure of a pallet of the present invention;

[0029] Figure 3 It is a structural schematic diagram of the head stage assembly of the double-layer fixed structure of the plate stage of the present invention;

[0030] Figure 4 is a global plan view of the skull obtained by image stitching of the present invention;

[0031] Figure 5 It is a coordinate system diagram established by the present invention;

[0032] Figure 6 This is a flow chart of the automated blind patch clamp operation of the present invention.

[0033] Figure 7 It is a waveform diagram of electrical signals successfully recorded in the experiment of the present invention.

[0034] In the figure, 1-a small hole for infiltrating dental cement to bond the skull; 2-an extension beam; 3-a screw; 4-a placement slot; 5-an electrode cleaning slot; 6-a high-toughness carbon fiber plate. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] The present invention suppresses the influence of animal head movement on patch clamp operation by designing and processing a double-layer fixing device for an animal head plate, and then accurately locates the craniotomy hole based on a global plane image of the skull, plans the path of the electrode into the brain based on coordinate transformation, and completes obstacle avoidance and sealing, membrane rupturing and signal recording of target cells based on electrode resistance feedback after entering the brain, and finally completes the automated blind patch clamp operation.

[0037] The present invention provides an animal automated blind patch clamp operation method, which belongs to the field of cell-level micromanipulation technology, and comprises the following steps: firstly, aiming at the interference problem of animal head movement on operation, a double-layer fixing device of an animal head plate is designed to reduce the influence of animal head movement on patch clamp operation; secondly, a global planar image of the skull is obtained by image stitching technology, and precise positioning of a craniotomy hole is completed based on the image; further, a movement path of an electrode entering a brain environment through a craniotomy hole is planned based on coordinate transformation; after the electrode enters the cerebral cortex, blood vessel obstacle avoidance and contact, high-resistance sealing and membrane rupture of a target cell are completed based on electrode resistance, and finally whole-cell electrophysiological signal recording is completed.

[0038] The present invention improves the stability of animal head fixation through the double-layer mechanical structure of the plate, accurately locates the craniotomy hole based on the global plane image of the skull, completes the microtube electrode trajectory planning according to the coordinate change, and finally realizes the blind patch clamp automation operation based on electrode resistance feedback. Compared with manual operation, the operation speed of this method is nearly doubled, the sealing success rate is increased by 5.8%, and the recording success rate is increased by 3.7%.

[0039] like Figure 1-Figure 6 As shown, the embodiment of the present invention discloses an animal-based blind patch clamp automated operating system, which is mainly used for experiments on 6-week-old C57BL / 6J mice, and specifically includes the following steps:

[0040] S101: Design and application of double-layer fixing device for animal head pallet

[0041] Before designing the fixture, the structure of the device was analyzed and divided into two parts: a head plate assembly and a head stage assembly. The head plate assembly was used to directly bond to the mouse skull to provide operating space and field of view. Combined with the brain conditions of 6-week-old mice used in the experiment, the head plate structure and size were preliminarily determined.

[0042] Since the mouse brain is conical and the exposed part of the skull after cutting off the scalp is elliptical, the interior of the head plate is designed as a hollow ellipse with a long axis of 12mm and a short axis of 8mm. The edge is set to a width of 3mm to ensure complete adhesion to the skull and reserve sufficient operating space for subsequent steps; four small holes 1 are designed at the edge for infiltrating dental cement to bond the skull, and the dental cement can be used to completely bond with the skull. The hollow part reserves space for patch clamp operation. In order to solve the problem of device stability after installing components, four extension beams 2 are designed for embedding and fixing with the head stage assembly. Compared with other types of mouse brain fixation devices, it can effectively prevent the edges from loosening.

[0043] The head stage assembly is used for the installation and support of the head plate assembly and is fixed to the experimental table. The head stage assembly is first designed with a placement groove 4 that matches the extension beam 2 of the head plate assembly. The depth of the groove is 3mm, which creates a height difference with the 5mm thick head plate assembly. At this time, screws 3 and high-toughness carbon fiber plates 6 are used in combination for fixing. The pressure generated by the bending of the high-toughness carbon fiber plates 6 is used to further stabilize the head plate assembly. The head stage assembly is additionally designed with 1.5cm*3cm rectangular electrode cleaning grooves 5 on both sides of the upper surface, which can be used for the placement and cleaning of electrodes.

[0044] Combining the experience of the artificial experimental process with actual problems, the material, size, groove depth and other parameters were selected, and the fixture was designed using Solidwords software. After completion, the device was installed on the experimental operation platform.

[0045] S102: Mouse craniotomy preparation

[0046] In this embodiment, the animal needs to be anesthetized and craniotomy performed before the experiment. The specific process is as follows:

[0047] (1) Female C57BL / 6J mice aged 6 weeks were taken from the animal center and anesthetized by intraperitoneal injection;

[0048] (2) The mouse was transferred to the operating table, the eyeball was covered with medical ointment, and the hair from the eye to the neck area of ​​the mouse was removed;

[0049] (3) Use surgical scissors and tweezers to cut the scalp and fascia from the eyes to the neck, and then wash and disinfect with 0.9% saline and 70% ethanol drops alternately;

[0050] (4) The mouse's head was fixed on a stereotaxic apparatus, and the skull was bonded to the head plate using dental cement and medical tissue glue;

[0051] (5) Use a 0.3 mm diameter cranial drill and 0.1 mm tip surgical forceps to perform craniotomy and remove the dura mater. Use 5°C 0.9% saline and hemostatic cotton to stop bleeding throughout the process.

[0052] (6) After craniotomy, the mouse was transferred to a blind patch clamp operation platform. After the fixation components were assembled, agarose prepared by 25 ml of cerebrospinal fluid with an osmotic pressure of 294-297 and 0.25 g of agar was dripped into the brain tissue in the cranial hole;

[0053] (7) Install electrodes and prepare for subsequent operations.

[0054] S2: Mouse skull image stitching and precise positioning of craniotomy holes based on image stitching.

[0055] In this embodiment, by controlling the platform to move back and forth in a zigzag pattern, m×n images of the skull microscopic field of view are collected and spliced ​​to form a global plane image of the skull including the craniotomy hole. The specific process is as follows:

[0056] (1) Focus the microscope on the lower left corner of the exposed skull image;

[0057] (2) The field of view is shifted along the x-axis in increments of 400 μm, and the CCD camera captures and records each field of view. After 16 shifts, these images are stitched together along the x-axis to form an image of the partially exposed skull;

[0058] (3) the view was shifted 800 μm along the negative y-axis and then along the negative x-axis to obtain the second stitched image;

[0059] (4) The above process is repeated 6 times to generate 12 preliminary stitched images;

[0060] (5) Cover the repeated portion to obtain a complete view of the exposed skull and clearly show the location of the skull opening.

[0061] After obtaining a complete skull view, the system automatically performs contour detection on the image to obtain the range and center point of the craniotomy hole. The threshold of contour detection is determined by the empirical value of the craniotomy hole size. The actual diameter of the craniotomy hole is approximately 0.5 mm.

[0062] S3: Coordinate system definition and electrode control

[0063] Define the following coordinate systems for the coordinates of the target point in different coordinate systems: Image coordinate system O i X i Y i Z i :Defined in the microscopic image captured by the CCD camera, the origin O i Located in the focal plane of the skull, plane X i O i Y i Focus view parallel to the exposed skull; skull coordinate system O s X s Y s Z s : Origin O s Defined as the focal point at the lower right corner of the focal plane exposing the skull. In this system, O i X i With O s X s Parallel, O i Y i With O s Y s parallel. So X s O s Y sThe plane is parallel to the skull focus view. The relationship between the skull coordinate system and the image coordinate system is determined as follows.

[0064]

[0065] Where m x ,m y ,m z Respectively represent the microscope along O i X i , O i Y i , O i Z i The displacement of s ,p i Represents the coordinates of point p in the image coordinate system and the skull coordinate system.

[0066] Electrode coordinate system O m X m Y m Z m : The origin is located at the tip of the electrode. i X i Y i Z i and O m X m Y m Z m Satisfies the following relationship:

[0067]

[0068] where p m represents the coordinates of point p in the electrode coordinate system. Therefore, point p can be represented in the skull coordinate system:

[0070]

[0071] According to the relationship between these three coordinate systems, the difference between the current position of the electrode tip and the coordinate value of the target point in the same coordinate system can be calculated, so that the electrode can be accurately guided to the target point on the cranial hole. Among them, the difference between the coordinates of the electrode tip and the target point on the X / Y axis can be calculated in the skull image plane, and the relative coordinate of the Z axis is calculated by the camera height difference Δh when the microscope focuses on the skull and the electrode tip respectively.

[0072] After obtaining the spatial coordinates of the electrode tip and the target point, the electrode is controlled to first descend along the Z axis until the relative coordinates of the two on the Z axis are 0, and then translated along the X / Y axis toward the target point. During the movement, the relative coordinates Δx and Δy of the two on the X / Y axis are calculated 3 times per second and the average is taken. When the average is within a reasonable error range, the electrode stops moving.

[0073] S4: Blind patch clamp automation process based on electrode resistance feedback:

[0074] The electrode is mounted on a microcontroller at a certain angle and moves obliquely downward at a fixed angle at a speed of 2 μm / s. If the electrode does not touch the neuron after reaching the preset maximum depth of 400 μm, it automatically withdraws from the brain tissue, indicating that the experiment has failed.

[0075] The electrode tip resistance is the only feedback in the operation process. The blind patch clamp operation can be divided into the following stages by the change of resistance value:

[0076] (1) After the electrode applies positive pressure and enters the brain tissue, the resistance value is 6 to 9 MΩ. While decreasing at a rate of 2 μm / s, the resistance value of the electrode tip is measured every 10 ms and the difference between the two adjacent resistance values ​​is calculated.

[0077] (2) If the difference between two consecutive recorded resistance values ​​is greater than 0.3 MΩ, the tip is considered to be close to the neuron, otherwise it continues to decrease.

[0078] (3) When the electrode contacts the neuron, the internal pressure is adjusted to atmospheric pressure. If the resistance increases significantly but does not exceed 100 MΩ, the internal pressure is switched to a low negative pressure of -10 hPa. Once the resistance exceeds 100 MΩ, the clamping voltage is switched to -70 mV to improve the sealing quality.

[0079] (4) When the resistance rises and stabilizes at the GΩ level, the sealing is considered successful; otherwise, the sealing fails and the neuron will fall off the electrode tip.

[0080] (5) After successful sealing, a series of short negative pressure pulses of -300 hPa are applied to the electrode to rupture the cell membrane of the neuron in contact with the tip, forming a whole-cell patch clamp structure; when the resistance drops below 500 MΩ and the discharge signal is detected, the membrane ruptures successfully and the signal begins to be recorded.

[0081] Among them, as the electrode enters the brain and gradually descends, the resistance of the electrode tip will increase when it contacts nerve cells and blood vessels. The speed of resistance increase can be used to judge and further control the electrode. When the electrode contacts nerve cells, the resistance increase is usually 0.3 to 0.5 MΩ; when the electrode contacts blood vessels, the increase is about 0.1 MΩ, and the electrode resistance will continue to increase by 0.1 MΩ as the electrode goes deeper. In this case, if the electrode continues to go deeper, the tip resistance value will eventually reach more than 20 MΩ.

[0082] Therefore, when it is judged that the electrode tip touches the blood vessel, obstacle avoidance control is required. Considering the angle of the electrode contacting the blood vessel and the diameter of the blood vessels in the brain of 6-week-old mice, the electrode is controlled to withdraw 10μm backward first, then move 10μm upward and continue to descend at 2μm per second to ensure that the electrode tip will not scratch the blood vessel and cause additional damage during the obstacle avoidance process. If it is found that the blood vessel is touched again during the descent after obstacle avoidance, it will directly withdraw from the brain tissue, otherwise it will continue to descend to complete the subsequent blind patch clamp operation.

[0083] According to the method of the present invention, 49 experiments were conducted on 6 mice. 14.3% (7 / 49) of the electrodes were blocked after being lowered to a certain depth; 45.2% (19 / 42) of the unblocked electrodes successfully established neuronal sealing, of which 42.1% (8 / 19) successfully completed recording.

[0084] Subsequent experimental results show that the success rate obtained by the method of the present invention is significantly improved compared with the sealing success rate of 39.4% (13 / 33) and the recording success rate of 38.4% (5 / 13) obtained by the manual method. The average operation time of the method of the present invention is 6 minutes and 10 seconds, which is 46% shorter than the average manual operation time of 11 minutes and 25 seconds.

[0085] The present invention is described in detail through embodiments, but the contents are only exemplary embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention; the protection scope of the present invention is defined by the claims, and any use of the technical solution described in the present invention, or any technicians in this field, inspired by the technical solution of the present invention, design a similar technical solution within the essence and protection scope of the present invention to achieve the above-mentioned technical effects, or any equal changes and improvements made to the scope of application, etc., should still fall within the scope of protection covered by the patent of the present invention.

Claims

1. An automated blind patch clamp operation method, characterized in that: The method comprises the following steps: S1: Design and manufacture a double-layer fixing device for the animal head platform to reduce the influence of animal head movement on the patch clamp operation, fix the animal head on the double-layer fixing device for the animal head platform, install electrodes and prepare for subsequent operations; S2: Obtain a global planar image of the skull through image stitching technology and accurately locate the craniotomy hole based on the image; S3: Planning the movement path of the electrode through the craniotomy hole into the brain environment based on coordinate transformation; S4: Based on the electrode resistance, the blood vessel is avoided and the target cells are contacted, sealed with high resistance and the membrane is broken, and finally the whole-cell electrophysiological signal recording is completed.

2. An automated blind patch clamp operation method according to claim 1, characterized in that: In S1, the double-layer fixing device for the animal head platform includes a two-layer structure of a head plate assembly and a head platform assembly. The head plate assembly is used to be bonded and connected to the animal's skull, and the head platform assembly supports the head plate assembly and is fixed to the platform.

3. An automated blind patch clamp operation method according to claim 2, characterized in that: The head plate assembly is designed with a hollow elliptical structure inside. The hollow elliptical structure is surrounded by small holes (1) for penetrating dental cement to bond the skull. The dental cement can completely bond the skull. The hollow part leaves space for patch clamp operation. Four extension beams (2) are provided between the head plate assembly and the head stage assembly.

4. The automated blind patch clamp operation method according to claim 3, characterized in that: The head stage assembly is provided with a placement groove (4) matching the extension beam. The head stage assembly is fixedly connected to the head plate assembly by screws (3) and an auxiliary high-toughness carbon fiber plate (6) for mounting and supporting the head plate assembly. An electrode cleaning groove (5) is respectively provided on both sides of the upper surface of the head stage assembly. The electrode cleaning groove (5) is used for cleaning operations of microtube electrodes.

5. The automated blind patch clamp operation method according to claim 1, characterized in that: In S2, the image stitching technology is specifically to collect m×n skull microscopic field images by controlling the platform to move back and forth in a zigzag shape, and stitch them to form a global plane image of the skull including the craniotomy hole.

6. The automated blind patch clamp operation method according to claim 1, characterized in that: In S2, after the images are stitched, the center and range of the craniotomy hole are obtained by automatic contour detection, and the threshold of contour detection is determined by the empirical value of the craniotomy hole size.

7. The automated blind patch clamp operation method according to claim 1, characterized in that: In S3, when planning the movement path of the electrode through the craniotomy hole into the brain environment, coordinate systems are established for the electrode tip, skull plane, and image plane respectively. The target position and the electrode tip are placed in the same coordinate system through coordinate transformation, so as to plan the movement path and control the movement of the electrode to the target position.

8. The automated blind patch clamp operation method according to claim 1, characterized in that: In S4, whether the electrode has entered the cerebral cortex is determined based on the electrode resistance value. After entering, it descends at a uniform speed and keeps the microtube at a slight positive pressure to prevent blockage of the tube mouth. When encountering an obstacle or a target cell, the microtube electrode resistance will increase significantly. The rising speed is used to determine whether the front end encounters the target nerve cell or blood vessel. If the rising speed of the resistance value is less than the threshold, it is considered that the blood vessel is encountered, and the electrode will first withdraw and then move laterally to avoid the blood vessel. If the rising speed of the electrode resistance value exceeds the threshold, it is considered that the target nerve cell is encountered, and the system automatically stops the microtube positive pressure, first applies a stable low negative pressure to complete the high-resistance sealing, and then applies a high negative pressure pulse to break the membrane, and finally completes the whole-cell electrophysiological signal recording.

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