A micromanipulation method for continuously performing a fixation injection and a sorting operation on the same target single cell

Abstract

The present application relates to the technical field of cell fixation injection and sorting, and discloses a micro-operation method for continuously performing fixation injection and sorting operations on the same target single cell. The present application can complete the fixation injection and sorting operations on the single cell, and the two continuous operations are performed on the same target single cell, thus avoiding many problems caused by switching different function operation needles in the conventional operation, such as confusing the target single cell with the remaining cells during the switching process of the injection and picking operations, or wasting time and effort in switching the operation needle, and reducing the risk of sample pollution and damage to the operation needle and the cell in the continuous and complex operation.

Description

Technical Field

[0001] This invention relates to the field of cell fixation, injection, and sorting technology, specifically a micromanipulation method for continuously fixing, injecting, and sorting the same target single cell. Background Technology

[0002] In practice, single-cell fixation-injection and sorting are often used separately, such as performing only fixation-injection or only sorting in a single single-cell operation. Currently, with the promotion and application of techniques such as single-cell micromanipulation, there is an increasing trend of successfully injected target single cells being directly sorted from uninjected single-cell populations for subsequent applications such as target single-cell cloning, single-cell PCR, single-cell sequencing, and single-cell mass spectrometry analysis. Thus, in a single experiment, it is necessary to continuously apply fixation-injection and sorting techniques to the same target single cell, using the same set of cell manipulation equipment (micromanipulation system including matching micromanipulation needles) to perform this series of operations continuously and complete the process.

[0003] However, in previous experiments, when switching between single-cell injection and pick-up operations, different single-cell micromanipulation needles had to be selected according to different operational requirements to meet different experimental needs. During the experimental operation, the operator had to change different micromanipulation needles according to actual needs. This switching process was time-consuming and labor-intensive. Moreover, during the switching process, successfully injected cells were often mixed with uninjected cells, making them difficult to distinguish, which increased the difficulty of this kind of experiment that required continuous injection and sorting, and even made it impossible to complete. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention aims to provide a micromanipulation method for continuously fixing, injecting, and sorting single cells of the same target. This method enables the use of a single-cell manipulation needle and a uniquely designed set of micromanipulation needles to perform direct sorting of single cells after injection, without altering the experimental conditions and equipment for single-cell injection and sorting. This eliminates the need for the existing method of switching between two sets of manipulation needles.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a micromanipulation method for continuously fixing, injecting, and sorting the same target single cell, comprising: Step 1: Measure the diameters of inner tube 1 and inner tube 2 in the inner tube of the fixed needle using a laser rangefinder, and record them as tube diameter 1 and tube diameter 2 respectively. Step 2: Take the unopened base material rod used to make the fixing pin, and use a drilling device to drill a hole in the base material rod so that the inner tube 2 is located in the middle of the inner tube 1, thereby changing the opening of the fixing pin from tube diameter 2 to tube diameter 1. Step 3: Install the newly made fixation needle and sorting needle symmetrically on the left and right sides of the micromanipulation device. The single cell first enters the inner tube 2 located in the middle through the opening of the fixation needle for fixation and injection. After the fixation and injection are completed, the sorting needle on the other side enters the fixation needle to sort the single cell. After sorting, the fixation needle ejects the single cell. This cycle is repeated to complete the continuous fixation, injection and sorting of single cells.

[0006] In some implementations, the original fixing pin mainly consists of an outer tube and an inner tube. The inner tube consists of two connected inner tubes, inner tube one and inner tube two, with different diameters. The diameter of inner tube two is smaller than that of inner tube one, and inner tube two is connected to the opening of the fixing pin.

[0007] In some embodiments, in the remade fixing pin, the inner tube is equivalent to being composed of two inner tubes one and one inner tube two connected together, with inner tube two located in the middle of the two inner tubes one, and one of the two inner tubes one communicating with the opening of the fixing pin.

[0008] In some implementations, the fixation needle controls the internal pressure of the needle tube, pushing the single cell to a designated location and ejecting it under positive pressure, and adsorbing and fixing the single cell under negative pressure.

[0009] In some embodiments, the micromanipulation device includes a fixation needle mounting mechanism, a sorting needle mounting mechanism, a pressure control mechanism, and a microscopic imaging mechanism. The pressure control mechanism is connected to the fixation needle and is used to control the formation of positive or negative pressure inside the fixation needle. The microscopic imaging mechanism is used to observe the process of single cells entering the fixation needle, fixation injection, sorting, and ejection in real time.

[0010] In some implementations, the length of the inner tube one located on one side of the fixation needle opening is shorter than the length of the inner tube one on the other side, so as to shorten the movement path of a single cell into the inner tube two and improve the efficiency of continuous fixation injection and sorting operations.

[0011] The technical solution provided by this invention has the following advantages compared with the prior art: This invention performs fixation, injection, and sorting of single cells simultaneously, both of which are performed on the same target single cell. This completely avoids many problems associated with switching between different functional needles in traditional methods, such as confusion between the target single cell and other cells during injection and pickup; or the time-consuming and laborious process of switching needles. It reduces the risk of sample contamination and needle and cell damage during these continuous and complex operations, avoids errors in continuous micromanipulation of the same target single cell, and improves the success rate of experiments. Ultimately, it achieves the goal of conveniently and quickly solving the above problems, promoting the smooth progress of related work. This technology can also adjust and record various parameters according to different target single cells, facilitating actual operation, improving experimental efficiency, and ensuring and improving the success rate of experiments. It is of great significance for current industry-academia-research innovation applications involving continuous fixation, injection, and sorting of the same target single cell. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the original fixing pin of the present invention; Figure 2 This is a schematic diagram of the new fixing pin of the present invention.

[0013] In the diagram: 1. Outer pipe; 2. Inner pipe; 201. Pipe 1; 202. Pipe 2. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] The original structure of the single-cell fixation needle is as follows: Figure 1 As shown, it mainly consists of an outer tube 1 and an inner tube 2. A single cell is fixed in the inner tube 202. After the injection is completed, the fixed, successfully injected cell needs to be ejected, the injection needle withdrawn from the corresponding device, and the sorting needle reinstalled and adjusted on the operating device before the sorting operation is performed on the same successfully injected single cell. This makes continuous operation impossible. To enable continuous fixation, injection, and sorting of the same target single cell, this invention provides a micromanipulation method for continuous fixation, injection, and sorting of the same target single cell, comprising: Remove the original fixing pin and measure the diameters of inner tube 1 201 and inner tube 2 202 in the inner tube 2 of the fixing pin using a laser rangefinder. These diameters are denoted as tube diameter 1 201 and tube diameter 2 202, respectively. Tube diameter 1 201 is larger than tube diameter 2 202.

[0016] Take an unopened base rod used to make the fixing pin. Based on the tube diameter 1 and tube diameter 2 obtained in the above steps, drill a hole in the base rod using a drilling device, so that the inner tube 202 is located in the middle of the inner tube 1 201, thereby changing the opening of the fixing pin from tube diameter 2 to tube diameter 1, as shown. Figure 2 As shown, in this case, the inner tube 2 can be regarded as consisting of two inner tubes 1 201 and one inner tube 2 202. The inner tube 2 202 is located between the two inner tubes 1 210. Furthermore, the length of the inner tube 1 210 that communicates with the opening of the fixation needle is less than the length of the other inner tube 1 202, so as to shorten the movement path of the single cell into the inner tube 2 202 and improve the efficiency of continuous fixation injection and sorting operations.

[0017] Third, the newly made fixation needles and sorting needles are symmetrically installed on the left and right sides of the micromanipulation equipment. The single cell first enters the inner tube 202 in the middle position through the opening of the fixation needle for fixation injection. Fixation is performed by adsorption fixation using negative pressure inside the needle tube. After the fixation injection is completed, the sorting needle on the other side enters the fixation needle to sort the single cell. After sorting, the fixation needle ejects the single cell through negative pressure. This cycle is repeated to complete the continuous fixation injection and sorting of single cells.

[0018] Experimental example: 1. Using a 100uL pipette and matching tip, prepare several single-cell injection operation and control droplets in the middle of a 60mm culture dish to prepare a single-cell injection dish; 2. Using a 100uL pipette and matching tip, prepare several single-cell sorting operation and control droplets in the middle of a 60mm culture dish to prepare a single-cell sorting dish; 3. First, place the single-cell injection dish on the microscope stage, and use a 10uL pipette and matching pipette tip to transfer the pre-prepared single cells into the pre-prepared operating droplet in the injection dish; 4. Adjust the microscope to clearly see the target single cell under the microscope; 5. Use a 10µL pipette and a micro-volume loading needle to load about 5µL of injection sample into the micromanipulation needle. After attaching the operating arm on one side of the micromanipulation system (usually the right side), adjust the needle tip to the blank droplet and perform a pre-injection to ensure that the injection needle is unobstructed and in normal condition. 6. After attaching the fixed sorting needle to the operating arm on one side of the micromanipulation system (usually the left side), lower it into the operating blank droplet to test the function of the micromanipulation instrument and the pneumatic microinjection instrument. Clean the head of the operating needle with the operating droplet. Finally, after the pressure is balanced, prepare for the formal experiment. 7. Operate the left-side micromanipulator and the matching pneumatic microinjector, select the target single cell to be manipulated, aspirate it into the fixation and sorting needle, and firmly fix the target single cell in the narrow part of the needle; 8. Operate the right-side micromanipulator to move the micromanipulation needle from the fixed sorting needle inlet to the fixed target single cell. After confirming that the injection position is correct, move it directly along the X-axis to accurately insert it into the target single cell. 9. Start the micro-automatic injection device and push the injection sample in the micromanipulation needle into the target single cell being processed. Here, you can see the fixed single cell surface under the microscope. Because there is sample inside, the outer membrane has obvious swelling. 10. Based on the microscopic observations, adjust the appropriate parameters of the components of the micro-operating system, including the movement speed and precise positioning memory of the operating instrument, the injection pressure and time parameters of the micro-automatic injector, and record them programmatically. Also, determine the optimal hand position for the pneumatic micro-injector (in this experiment, the micro-automatic injector parameters are set as follows: injection pressure 246 hPa, injection time 0.2 seconds, equilibrium pressure 20 hPa; the injection pressure action is generated after pressing the foot pedal, and the injection pressure automatically stops after the desired effect is achieved). If the injection results are satisfactory, the complete set of injection operation parameters can be stored through the program recording function. 11. After injecting the target single cell, first withdraw the injection needle, then keep the single cell firmly fixed in the fixed sorting needle tube, and directly perform the next sorting operation on the same target single cell. 12. Carefully remove the injection droplet from the fixation and sorting needle containing the target single cell at the needle tip. 13. Remove the single-cell injection dish from the microscope stage and replace it with a single-cell sorting dish; 14. Adjust the microscope to clearly see the edges of the sorted droplets under the microscope; 15. Drive the corresponding operating arm to carefully move the fixed sorting needle containing the target single cell into the sorting droplet and observe whether the fixed target single cell in the needle tube is in a normal state. 16. Carefully release the negative pressure inside the hydraulic microinjector to release the target single cell from the fixation and sorting needle into the sorting droplet. Finally, withdraw the instrument and the corresponding single cell fixation, injection, and sorting needles. For the same target single cell, the fixation, injection, and sorting operations are completed continuously. The separated single cell samples can be processed in various ways, such as single-cell PCR, single-cell sequencing, single-cell cloning, and other operations.

[0019] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. The computer-readable medium described above in this application can be a computer-readable signal medium or a computer-readable storage medium, or any combination of the two. A computer-readable storage medium can be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination thereof. In this application, a computer-readable storage medium can be any tangible medium that contains or stores a program that can be used by or in conjunction with an instruction execution system, apparatus, or device. In this application, a computer-readable signal medium can include a data signal propagated in baseband or as part of a carrier wave, carrying computer-readable program code.

[0020] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A micromanipulation method for continuously fixing, injecting, and sorting single cells of the same target, characterized in that, include: Step 1: Measure the diameters of inner tube 1 and inner tube 2 in the inner tube of the fixed needle using a laser rangefinder, and record them as tube diameter 1 and tube diameter 2 respectively. Step 2: Take the unopened base material rod used to make the fixing pin, and use a drilling device to drill a hole in the base material rod so that the inner tube 2 is located in the middle of the inner tube 1, thereby changing the opening of the fixing pin from tube diameter 2 to tube diameter 1. Step 3: Install the newly made fixation needle and sorting needle symmetrically on the left and right sides of the micromanipulation device. The single cell first enters the inner tube 2 located in the middle through the opening of the fixation needle for fixation and injection. After the fixation and injection are completed, the sorting needle on the other side enters the fixation needle to sort the single cell. After sorting, the fixation needle ejects the single cell. This cycle is repeated to complete the continuous fixation, injection and sorting of single cells.

2. The micromanipulation method for continuously fixing, injecting, and sorting the same target single cell according to claim 1, characterized in that, The original fixing pin mainly consists of an outer tube and an inner tube. The inner tube is composed of two connected inner tubes, inner tube one and inner tube two, with different diameters. The diameter of inner tube two is smaller than that of inner tube one, and inner tube two is connected to the opening of the fixing pin.

3. The micromanipulation method for continuously fixing, injecting, and sorting the same target single cell according to claim 1, characterized in that, In the remade fixing pin, the inner tube is equivalent to being composed of two inner tubes one and one inner tube two connected together. The inner tube two is located in the middle of the two inner tubes one, and one of the two inner tubes one is connected to the opening of the fixing pin.

4. The micromanipulation method for continuously fixing, injecting, and sorting the same target single cell according to claim 1, characterized in that, The fixation needle controls the internal pressure of the needle tube. Under positive pressure, it pushes the single cell to a designated location to eject the single cell, and under negative pressure, it adsorbs and fixes the single cell.

5. The micromanipulation method for continuously fixing, injecting, and sorting the same target single cell according to claim 1, characterized in that, The micromanipulation device includes a fixation needle mounting mechanism, a sorting needle mounting mechanism, a pressure control mechanism, and a microscopic imaging mechanism. The pressure control mechanism is connected to the fixation needle and is used to control the formation of positive or negative pressure inside the fixation needle. The microscopic imaging mechanism is used to observe the process of single cells entering the fixation needle, fixation injection, sorting, and ejection in real time.

6. The micromanipulation method for continuously fixing, injecting, and sorting the same target single cell according to claim 3, characterized in that, The length of the inner tube one located on one side of the fixation needle opening is shorter than the length of the inner tube one on the other side, so as to shorten the movement path of single cells into the inner tube two and improve the efficiency of continuous fixation injection and sorting operations.

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