JP2008271804A - Method and system for microinjection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for injecting a substance into a desired site in a cell by automatically and accurately detecting the tip position of a pipette for an individual cell and stopping the pipette at a target position in the cell. <P>SOLUTION: The method comprises: a first step of injecting one cell 31 with a barrel pipette 10 equipped with voltage-measuring electrodes 11 at a predetermined speed and measuring the voltage at a cell site where the tip of the barrel pipette 10 is situated; a second step of differentiating a signal representing thus measured voltage, and when the peak of the differentiated signal exceeds a threshold value, judging that the tip of the barrel pipette is inserted into an intracellular small organ (organelle), stopping the movement of a manipulator when the tip of the barrel pipette comes to a desired site, and injecting a reagent into the organelle. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microinjection method and a microinjection apparatus capable of automatic insertion for introducing a reagent into a cell.

  In the field of biotechnology, by introducing genes, proteins, various inhibitors, molecular targeting drugs, etc. into cells and organelles (intracellular organelles) such as the nucleus, mitochondria, and Golgi apparatus There is an increasing demand for evaluating expression and behavior in cells.

  As a method for injecting a substance into a cell, there is known a microinjection method in which a hollow pipette processed by heating and stretching a capillary made of glass or quartz is directly injected into a cell. . Although the microinjection method is recognized as a useful method for directly injecting various bioactive substances into target cells, it is necessary for a skilled operator to operate the micromanipulator while looking through a microscope. Only a few hundred cells can be processed per day. As a method for automatically performing microinjection, Anal. Chem., 77, 5628 (2005) proposes a method of fixing a cell in a microwell using a plate having a large number of microwells.

Anal. Chem., 77, 5628 (2005)

  In the cell, compartments surrounded by a very thin layer called a lipid bilayer, such as a cell membrane, are arranged three-dimensionally and are called organelles. Among these are the nucleus, mitochondria, Golgi apparatus, vacuole, etc., which are configured as a compartment different from the cytoplasm. Recently, it has been known that local introduction into the cytoplasm and organelle is necessary even within the cell, depending on the type of biologically active substance.

  In the case of a method for determining the insertion coordinate position of a cell using a plate having a microwell, the tip of a pipette for injecting a bioactive substance is driven to XY coordinates representing the center position of the well. When a certain amount of manipulator is operated using only the XY coordinates as an index and a pipette is inserted, the pipette penetrates the cell wall or cell membrane and then reaches the vicinity of the center of the cell. Therefore, depending on the type of biologically active substance, it is impossible to locally introduce and sort into the cytoplasm and organelle within the cell, and most cells die. This indicates that the penetration depth of the pipette is desirably as shallow as possible in the local structure. Therefore, it is necessary to stop the movement of the pipette immediately after the tip of the pipette penetrates the cell membrane or the organelle membrane. This stop position needs to be determined with an accuracy of 0.5 μm. However, since the cells are different in size, it is difficult to determine the XY coordinates that are the movement targets of the pipette based on the position of the well. Furthermore, when the pipette pushes through the cell membrane, the cell size changes due to deformation. Further, as described above, depending on the purpose such as evaluation of a medicine, it is necessary to locally aim at each of these locations and introduce the medicine. Therefore, there is a need for a mechanism for stopping the movement of the microinjection pipette by a stop signal generated based on another principle that does not depend on the cell size.

  The present invention provides a method for automatically and accurately detecting the tip position of a pipette with respect to an individual cell, stopping the pipette at a target position in the cell, and a method for introducing a substance into a desired position in the cell. The purpose is to provide.

  When the microelectrode penetrates the cell membrane, the membrane potential changes rapidly. For living cells, this potential is negative with respect to the external medium. We considered using the change in membrane potential as a stop signal.

  FIG. 1 is a diagram schematically showing a change in potential at a barrel pipette tip position when a barrel pipette is inserted into a cell. The pattern of potential change that occurs each time the cell membrane or organelle membrane is penetrated varies depending on the electrolyte concentration in the cytoplasm or organelle, but in living cells, the potential in the cytoplasm is negative from the outside. The potential in the organelle varies depending on the type and physiological state of the organelle. For example, in the case of a vacuole, the potential is usually on the positive side of the cytoplasm. Reference numerals in FIGS. 1A, 1B, and 1C correspond to the positions of the pipettes shown in FIG. FIG. 1A shows potential changes that occur as the barrel pipette advances in the order of extracellular (A1), cell membrane vicinity (A2), cytoplasm (A3), and vacuole (A4). FIG. 1B shows a case where the potential in the organelle 1 is a potential on the negative side of the cytoplasm. FIG. 1 (c) shows a potential change in the case where the organelle 2 has a potential more negative than the cell potential and the vacuole has a potential more positive than the cytoplasm. In this way, although the level of the membrane potential is different, it can be determined in real time that it has been inserted into the cytoplasm or organelle if the potential change pattern accompanying the movement of the barrel pipette is accumulated, and it is highly useful .

  The microinjection method capable of automatic insertion according to the present invention includes a step of positioning a tip of a barrel pipette as a microtool having a potential measuring electrode with respect to one cell, and a barrel pipette with respect to one cell. Moving at a speed, inserting the tip into the cell, measuring the potential of the cell site where the tip of the barrel pipette is located by the potential measuring electrode, and changing the potential of the barrel pipette from the change in potential accompanying the movement of the barrel pipette. A step of stopping the movement of the barrel pipette when it is determined that the tip has reached a desired site in the cell, and a step of injecting a reagent from the barrel pipette. In determining whether or not the tip of the barrel pipette has reached a desired site in the cell, it can be determined that the tip of the barrel pipette has been inserted into another organelle when the measured potential signal changes suddenly.

  The microinjection apparatus capable of automatic insertion according to the present invention includes a manipulator including a reagent pipette filled with a solution containing a potential measuring electrode and a reagent, a driving unit for driving the manipulator, and a movement of a barrel pipette as a micro tool. A signal processing unit that obtains information about a cell site where the tip of the barrel pipette is located from the output change of the potential measurement electrode, a control unit that controls the driving unit by the output of the signal processing unit, and a solution that contains the reagent from the reagent pipette And an injector for ejecting the fuel. The signal processing unit includes a low-pass filter to which the output of the potential measurement electrode is input and a differentiation circuit that differentiates the output of the low-pass filter, and the output of the potential measurement electrode that has been differentiated by the differentiation circuit exceeds a predetermined threshold value. In some cases, a signal indicating that the tip of the barrel pipette has been inserted into the organelle can be output. The potential measuring electrode can be composed of a pipette filled with a liquid or solid conductive substance.

  In the present invention, it is important to be able to stop the automatic insertion of the barrel pipette and keep the tip of the barrel pipette at the target site in the cell. Thereafter, if a reagent injection signal is applied to the barrel pipette, the reagent can be introduced into a desired site in the cell.

  According to the present invention, the position of the tip of a barrel pipette in a cell can be accurately determined, and a gene or drug can be reliably introduced into a desired site in an individual cell without killing the cell.

  Embodiments of the present invention will be described below with reference to the drawings. In the following, the case of FIG. 1 (a), that is, the case of selecting and inserting into the cytoplasm of a plant cell or a vacuole will be mainly described as an example.

  FIG. 2 is a schematic view showing an example of a microinjection apparatus capable of automatic insertion according to the present invention. This apparatus is for introducing reagents such as genes, proteins, various inhibitors, and molecular target drugs from a barrel pipette 10 as a micro tool into cells 31 in a solution placed in a container 30. The barrel pipette 10 is a three-channel barrel pipette having a tip diameter of about 1 μm. One channel of the barrel pipette is filled with 0.5 M KCl solution as an electrolyte, and an Ag / AgCl wire is inserted and used as the potential measuring electrode 11. The other channel is filled with 0.5 M KCl and a reagent to be introduced into the cell, and a Pt wire is inserted and used as the reagent injection electrode 12. The remaining channel is filled with 0.5 M KCl solution as an electrolyte, and a Pt wire is inserted and used as the counter electrode 13 for the reagent injection electrode 12. The introduction of the reagent injection electrode 12 can be confirmed by mixing a fluorescent dye as a tracer. As a reference electrode 21 for the potential measuring electrode 11, a commercially available Ag / AgCl electrode is immersed in the solution in the container 30.

  The potential measuring electrode 11 and the reference electrode 21 are connected to the signal processing unit 22. The output of the signal processing unit 22 is supplied to the overall control unit 29. The overall control unit 29 is equipped with an automatic operation / manual operation switching switch. In manual operation, every time the switch is clicked, a signal for driving the pulse motor is output for 0.1 seconds. In the automatic operation, the motor drive control unit 23 continuously drives and controls the pulse motor 24 directly connected to the hydraulic manipulator 25 until the stop signal is output from the signal processing unit 22 from the drive start signal. When the hydraulic manipulator 25 rotates clockwise, the Z-axis insertion manipulator 50 advances, and the barrel pipette 10 connected to the tip of the Z-axis insertion manipulator advances accordingly. The minimum step of the Z-axis insertion manipulator 50 is 0.5 μm, and the maximum moving speed is 250 μm / second. The reagent injection electrode 12 and the counter electrode 13 are connected to a reagent injection signal generator 27. The reagent injection signal generator 27 generates a reagent injection pulse under the control of the overall control unit 29. After an analog rectangular pulse is generated from the pulse generator and amplified, a pulse voltage of −20 V is applied to the reagent injection electrode 12 with respect to the counter electrode 13. As an example, the reagent injection pulse has a pulse width of 10-50 ms and a pulse number of 1-5 pulses at intervals of 1 second. The motor drive control unit 23 is also controlled by the overall control unit 29. A manual operation dial 26 is provided between the pulse motor 24 and the hydraulic manipulator 25. A recorder 28 is connected to the signal processing unit 22.

  FIG. 3 is a block diagram showing details of signal flow and signal processing in the microinjection apparatus capable of automatic insertion according to the present invention. The signal processing unit includes an amplifier, a low-pass filter, a differentiation circuit, and a discriminator. The output of the potential measurement electrode with respect to the reference electrode is amplified by an amplifier and then passed through a low-pass filter. The signal that has passed through the low-pass filter is differentiated by a differentiation circuit and passed to a discriminator. The discriminator of this example includes a non-inverting amplifier circuit, an inverting amplifier, two half-wave rectifiers, and two comparators. The signal processing unit in this example generates a cytoplasm signal and a vacuole signal, and the cytoplasm signal and the vacuole signal are supplied to the overall control unit 29. The overall control unit 29 controls the motor drive control unit 23 and the reagent injection signal generator 27.

  In this example, voltage application was used as a method for injecting the reagent in the pipette into the cells, so the pipette filled with the reagent was called a reagent injection electrode, but other methods such as pressure application were used as the reagent injection method. May be. In the case of injecting a reagent by applying pressure, the reagent injection signal generator 27 applies a pressure pulse instead of an electric pulse to the rear part of the pipette filled with the reagent. Of course, the Pt wire inserted into the pipette filled with the reagent in the above example is unnecessary, and the counter electrode 13 is also unnecessary.

  Next, an automatic insertion microinjection method using the apparatus of the present invention will be described. The cell used for the experiment is tobacco cultured cell BY-2. Usually, for drug introduction / transformation into plant cells, it is common to use a soft protoplast or spheroplast to treat the cell surface with an enzyme, and to intact plant cells while maintaining the cell wall. Although continuous injection has been considered difficult, the apparatus of the present invention makes it possible.

  Initially, the barrel pipette 10 was positioned near one cell 31 in the container 30 by a semi-automatic operation while observing under a microscope. Next, in order to obtain the space potential distribution, the barrel pipette 10 was advanced 100 ms at a speed of 250 μm / sec. When the barrel pipette 10 penetrated the cell membrane, a noisy response was obtained from the potential measuring electrode 11.

FIG. 4 shows an output signal and a differential signal obtained when the insertion process is performed by manual operation. In the signal processing unit 22, the signal from the potential measurement electrode 11 that has passed through the low-pass filter is differentiated by a differentiation circuit. FIG. 4A shows a typical example of the signal from the potential measuring electrode 11 after passing through the low-pass filter (amplified by 10 times). In step A in the figure, the barrel pipette 10 was brought close to the outer surface of the cell membrane by manual operation. In Step B and Step D, the control switch was clicked once (250 μm / second × 100 ms). In Steps C and E, the Z-axis insertion manipulator was stopped. FIG. 4B shows a signal amplified after differentiating the signal from the potential measurement electrode 11 that has passed through the low-pass filter. By differentiating the output of the potential measuring electrode, a positive pulse T ₁ and a negative pulse T ₂ are obtained as shown in FIG. The positive pulse T1, which is the first peak, is a signal penetrating the cell membrane from the outside. As shown in FIG. 4 (b), when the positive pulse T1, which is the first peak of about 200 mV, was actually obtained, the insertion was stopped and the fluorescent dye was injected into the protoplasm. The negative pulse T2 which is the second peak is a signal penetrating the vacuolar membrane. Here, after the positive pulse T1 as the first peak is obtained, the insertion is further continued, and both the negative pulse T2 as the second peak is obtained as shown in FIG. 4B. When inserted, the insertion was stopped, and when the fluorescent dye was injected, it was introduced into the vacuole. These pulse signals are non-inverted or inverted and input to the discriminator. Thus, a cytoplasm signal and a vacuole signal are obtained.

FIG. 5 is a schematic diagram showing the relationship between the signal waveform processed by the discriminator of the signal processing unit, the cytoplasm signal, and the vacuole signal. A cytoplasm signal is output when the positive pulse T ₁ included in the differential signal exceeds a preset threshold, and a vacuole signal is output when the negative pulse T ₂ exceeds a preset threshold. Upon receiving these signals, the overall control unit 29 instructs the motor drive control unit 23 to stop driving the pulse motor 24. That is, when the barrel pipette 10 needs to be stopped in the cytoplasm, the overall control unit 29 issues a pulse motor stop command to the motor drive control unit 23 when a cytoplasm signal is output from the signal processing unit. When it is necessary to stop the barrel pipette 10 within the vacuole, the overall control unit 29 issues a pulse motor stop command to the motor drive control unit 23 when the vacuole signal is output from the signal processing unit.

  FIG. 6 is a control flowchart of the motor. In the case of the present embodiment, two operation modes are prepared: a cytoplasmic stop mode and a vacuole stop mode. The operation mode is selected by the overall control unit 29. The motor drive control unit 23 drives the step motor 24 to advance the barrel pipette 10 by 500 nm at a time, and determines whether a cytoplasm signal or a vacuole signal has been detected in accordance with the selected operation mode. If it is not detected, the barrel pipette 10 is again advanced by 500 nm. When the desired signal is detected, the motor is stopped. The motor drive pulse is turned on / off by the motor drive control unit 23, and the detection determination of the cytoplasm signal or the vacuole signal is performed by the overall control unit 29.

  The overall control unit 29 can instruct the motor drive control unit 23 to insert (forward), retract (retreat), stop, and quickly retract (rapid retract) the barrel pipette 10. The overall control unit 29 acquires information related to the tip position of the barrel pipette 10 based on a signal from the signal processing unit 22, and automatically sets the tip of the barrel pipette with respect to the cell by setting an arbitrary position in the cell. It can be driven and stopped at any position. After receiving a signal from the signal processing unit 22 indicating that the tip of the barrel pipette 10 has reached a desired position in the cell, the overall control unit 29 instructs the reagent injection signal generator 27 to inject the reagent of the barrel pipette. A reagent is introduced from the tip of the electrode 12 to a desired site in the cell. The introduction amount of the reagent can also be set from the overall control unit 29, and the reagent injection signal generator 27 generates a reagent injection pulse having a pulse width, an interval time, and the number of times determined according to the setting. Thus, by applying a voltage between the reagent injection electrode 12 and the counter electrode 13 and applying an introduction load, the drug in the reagent injection electrode 12 is injected into the cell. In order to introduce a reagent into a cell, it cannot be achieved simply by inserting the barrel pipette 10, and it is necessary to apply such an introduction load.

  In automatic operation, the barrel pipette was slowly advanced at 4 μm / sec in order to shorten the braking distance after detection of the stop signal. At this speed, a smooth signal waveform was obtained as shown in FIGS.

  FIG. 7 is a diagram showing the measurement result of the potential change obtained when the barrel pipette is inserted into the cytoplasm and the result of waveform conversion by the differentiation circuit. FIG. 7A is a waveform diagram showing an amplified output of the potential measurement electrode when the tip of the barrel pipette is inserted from the vicinity of the cell membrane into the intracellular (cytoplasmic) direction. FIG. 7B shows the differential signal.

  After the tip of the barrel pipette contacts the cell membrane, it reaches the cytoplasm when further inserted. At this time, the potential change is inclined in the negative direction by contacting a film having a negative charge from the positive direction. This is shown in FIG. 7 (a). When this raw data is always taken and passed through a differentiating circuit, it is converted as shown in FIG. FIG. 7C is a diagram showing a state of drive control of the Z-axis insertion manipulator, in which the solid color represents the forward movement, and the white portion represents the state stopped after the detection of the cytoplasm signal.

  FIG. 8 is a diagram showing the measurement result of the potential change obtained when the barrel pipette is inserted into the vacuole which is a kind of organelle through the cytoplasm, and the result of waveform conversion by the differentiation circuit. FIG. 8A is a waveform diagram illustrating the output of the potential measurement electrode after amplification. FIG. 8B shows the differential signal.

  When the tip of the barrel pipette is inserted from the vicinity of the cell membrane into the cell (cytoplasm) direction, it contacts the cell membrane, and reaches the cytoplasm when further inserted. At this time, the potential change is inclined in the negative direction by contacting a film having a negative charge from the positive direction. This is the same as in the case of FIG. When inserted further, it passes through the membrane of an organelle (such as a vacuole) isolated by a lipid bilayer in the center of the cell, and reaches the organelle. This is shown in FIG. 8 (a). Further, when this raw data is always taken and passed through a differentiating circuit, it is converted as shown in FIG. FIG. 8C is a diagram showing a state of drive control of the Z-axis insertion manipulator, in which the solid portion represents the forward movement, and the white portion represents the state stopped after the detection of the cytoplasm signal.

  Whether the automatic insertion microinjection by the apparatus of the present invention was successful was evaluated by microscopic observation. Lucifer yellow was added as a fluorescent reagent to the reagent injection electrode, and the reagent was locally introduced into a desired site in the cell. The cell used was tobacco cultured cell BY-2.

  FIG. 9 shows a case where the tip of the barrel pipette is automatically stopped in the cytoplasm, a pulse signal is applied to the reagent injection electrode to inject lucifer yellow, and then the diffusion pattern of lucifer yellow is observed with a fluorescence microscope. FIG. 9 (a) shows an operation in which the barrel pipette is automatically stopped in the cytoplasm, and then a pulse signal is applied to the reagent injection electrode to continuously inject lucifer yellow and is positioned at the center of FIG. 9 (a). It is the relief contrast image (bright field) of the state stopped automatically in the cytoplasm of the cell to do. The bar size shown in the figure is 20 μm. FIG. 9B is a fluorescence image after lucifer yellow is introduced into the cells. According to FIG. 9B, the fluorescence emission region is localized in the cell, and it can be confirmed that lucifer yellow has been introduced into the cytoplasm of the cell located in the center.

  Next, after the barrel pipette tip is automatically stopped in the vacuole of cultured tobacco cell BY-2, lucifer yellow is injected by applying a pulse signal to the reagent injection electrode, and then the diffusion pattern of lucifer yellow is observed with a fluorescence microscope. Was observed. FIG. 10A is a relief contrast image after the barrel pipette tip is automatically stopped in the vacuole of the cell located at the center. FIG. 10B is a fluorescence image after lucifer yellow is introduced into the vacuole of the cell located in the center. According to the fluorescence image of FIG. 10B, it can be confirmed that fluorescence is generated from a wide area in the cell, the tip of the barrel pipette stops in the vacuole, and lucifer yellow is introduced into the vacuole.

  Although the example of the plant cell with a cell wall was demonstrated so far, this invention is applicable also to the animal cell etc. which have no cell wall and are covered with the cell membrane. FIG. 11A shows a differential output signal of the potential measurement electrode when a barrel pipette is inserted into a mouse embryonic stem cell as an example of an animal cell. FIG. 11B shows a differential output signal of the potential measurement electrode for a rice cell in a state where the cell wall is removed from the plant cell which originally has a cell wall through the enzyme treatment and the cell membrane is exposed (protoplast). Each cell type shows a different pattern, but all show rapid potential changes when penetrating the cell membrane, applying the principle of the present invention to stop the barrel pipette at the desired position in the cell and introduce the reagent. You can see that it is possible.

  Thus, it was found that the electric potential showed a specific pattern as the barrel pipette moved inside the cell. As described above, intracellular organelles include nuclei, endoplasmic reticulum, mitochondria, vacuoles, etc., and mitochondria are known to have a relatively high potential. Each has the characteristic of being separated from the outside by a lipid bilayer membrane, and although the membrane potential level is different, if the potential change pattern accompanying the movement of the barrel pipette is accumulated, each organelle can be accumulated. Can be determined, and is highly useful. For example, as a specific organelle, it is possible to inject a drug only into the nucleus. For this reason, it becomes possible to actively cause high-efficiency incorporation of DNA, and industrial merit is high.

  As described above, according to the present invention, it is possible to freely set the reagent injection site in the cell, and the reagent injection can be automatically performed. Such a thing is difficult with the conventional technology, and just shows the usefulness of the present invention.

  Comparing the transfection method with the single cell introduction method according to the present invention, the transfection method can obtain a certain percentage of cells into which the gene has been introduced, but in order to increase the ratio of the transfected cells to nearly 100%, It is necessary to perform subculture and cloning in the presence of selection pressure for several months. On the other hand, since the introduction into a single cell of the present invention literally targets one cell, if the introduced cell is isolated and cloned, sampling can be performed when the number of cells increases to the extent of analysis. Therefore, the required time can be greatly shortened. Real-time PCR analysis can be performed from a single cell, and it can save a lot of labor, time, and equipment, so it can be an unprecedented new experimental technique. It is done.

The figure which shows typically the electrical potential change in a barrel pipette tip position when a barrel pipette is inserted in the cell. The schematic diagram which shows an example of the microinjection apparatus by this invention. FIG. 3 is a block diagram showing details of signal flow and signal processing in the microinjection apparatus of the present invention. The figure which shows the example of the signal from an electric potential measurement electrode, and its differential signal. The schematic diagram showing the relationship between the signal waveform processed with the discriminator of a signal processing part, a cytoplasm signal, and a vacuole signal. FIG. The figure which shows the measurement result of the electrical potential change obtained when a barrel pipette is inserted in cytoplasm, and the result of having performed the waveform conversion by a differentiation circuit. The figure which shows the measurement result of the electrical potential change obtained when a barrel pipette is inserted in a vacuole, and the result of having performed the waveform conversion by a differentiation circuit. The figure which shows the example which inject | poured the fluorescence reagent in cytoplasm and observed with the fluorescence microscope. The figure which shows the example which inject | poured the fluorescence reagent in the vacuole and observed with the fluorescence microscope. The figure which shows the differential output signal pattern when inserting in the plant cell which removed the embryonic stem cell and cell wall of the animal.

Explanation of symbols

10: barrel pipette 11: potential measuring electrode 12: reagent injection electrode 13: counter electrode 21: reference electrode 22: signal processing unit 23: motor drive control unit 24: pulse motor 25: hydraulic manipulator 26: manual operation dial 27: reagent Injection signal generator 28: recorder 29: overall control unit 30: container 31: cell 50: Z-axis insertion manipulator

Claims (10)

Positioning the tip of a barrel pipette with a potential measuring electrode relative to one cell;
Moving the barrel pipette with respect to the cell at a predetermined speed, and inserting a tip into the cell;
Measuring the potential of the cell site where the tip of the barrel pipette is located by the potential measuring electrode;
Stopping the movement of the barrel pipette when it is determined that the tip of the barrel pipette has reached a desired site in the cell from the change in potential associated with the movement of the barrel pipette;
And a step of injecting a reagent from the barrel pipette.   2. The microinjection method according to claim 1, wherein the signal representing the potential is differentiated, and the movement of the barrel pipette is stopped when the differential signal suddenly changes.   The microinjection method according to claim 1 or 2, wherein the step of moving the barrel pipette at a predetermined speed and the step of measuring the potential until it is determined that the tip of the barrel pipette has reached a desired site in the cell. A microinjection method characterized by repeatedly executing   4. The microinjection method according to claim 1, wherein the potential measuring electrode is a pipette filled with a liquid or solid conductive material. A manipulator comprising a reagent pipette filled with a solution containing a potential measuring electrode and a reagent;
A drive unit for driving the manipulator;
A signal processing unit for acquiring information on a cell site where a tip of the barrel pipette is located from an output change of the potential measuring electrode accompanying the movement of the barrel pipette;
A control unit that controls the driving unit based on an output of the signal processing unit;
A microinjection apparatus comprising an injector for ejecting a solution containing a reagent from the reagent pipette.   6. The microinjection device according to claim 5, wherein the potential measuring electrode is a pipette filled with an electrolyte.   The microinjection device according to claim 5 or 6, wherein the signal processing unit includes a low-pass filter to which an output of the potential measurement electrode is input and a differentiation circuit that differentiates the output of the low-pass filter, and the output of the differentiation circuit is A microinjection apparatus that outputs a signal when a predetermined threshold value is exceeded.   8. The microinjection device according to claim 7, wherein when the signal processing unit outputs a signal, the control unit stops the movement of the manipulator by the driving unit, and drives the injector to drive a reagent from the reagent pipette. A microinjection apparatus characterized by ejecting a solution containing   The microinjection apparatus according to any one of claims 5 to 8, further comprising a reference electrode immersed in a solution containing cells.   10. The microinjection apparatus according to claim 5, wherein the ejection unit is a unit that applies a pulse voltage to the reagent pipette. 11.

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