A method for measuring intracellular pressure based on microtube electrode resistance

Abstract

The application provides a microtubule electrode resistance-based intracellular pressure measuring method, and belongs to the technical field of cell operation, and comprises the following steps: 1, after the microtubule electrode pierces into a cell, the pressure of the injection gas pressure of the microtubule electrode is continuously increased, and the resistance value of the microtubule electrode is measured in real time until a quasi-stable state is reached, and the injection gas pressure P1 at this time is recorded; 2, the microtubule electrode is completely withdrawn from the cell, and the intracellular pressure is released through the wound; 3, the microtubule electrode is controlled to pierce into the cell along the same track as the first time, and the injection gas pressure P2 is obtained by using the same method as in step 1; 4, the difference between P1 and P2 is the intracellular pressure value. The application can use common electrode resistance measuring equipment, and does not need a micro force sensor and a special equipment with a closed-loop gas pressure rapid adjusting function; after an operator moves a suction needle and an injection needle into a field of view, the remaining steps can be automatically completed, and the measuring result is less affected by the professional level of the operator.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell operation, and particularly relates to a method for measuring intracellular pressure based on microtube electrode resistance. BACKGROUND

[0002] Intracellular pressure is an important parameter of intracellular environment, and has important regulating effects on normal physiological activities of cells and development process of embryos. By measuring intracellular pressure, intracellular environment mechanism of physiological activities of cells and embryo development can be explored. At present, the methods for measuring intracellular pressure mainly include the following three ways: measuring by injecting oil droplet volume, measuring based on elastic change of cells before and after membrane rupture, and measuring based on resistance change of micro-needle before and after puncture. Among them: the method of injecting oil droplets has greater damage to intracellular environment; the method based on elastic change of cells before and after membrane rupture needs to be directly connected with a high-precision micro-force sensor, and this measurement method poses a series of challenges to the design of micro-needle gas circuit; in addition, the intracellular pressure detection method based on electrical properties of microtubes before and after puncture needs to be equipped with an expensive microtube resistance detection device with a function of automatically adjusting pressure quickly, so that the measurement is more complex and the cost is higher. Therefore, it is necessary to develop a method for measuring intracellular pressure based on conventional resistance detection device, which is non-invasive to cells, simple and reliable in measurement system, and low in measurement cost. SUMMARY

[0003] The present application provides a method for measuring intracellular pressure based on microtube electrode resistance, which detects intracellular pressure by the difference of injection gas pressure required by twice intracellular injection in quasi-equilibrium state before and after intracellular pressure release, and can be realized on conventional electrode resistance detection equipment such as general patch clamp equipment.

[0004] The technical scheme adopted by the present application is: a method for measuring intracellular pressure based on microtube electrode resistance, comprising the following steps:

[0005] Step 1: the cell is fixed by suctioning a microtube; after the microtube electrode is punctured into the cell, the microtube electrode is retreated to a position where the extrusion shape of the cell is restored; the injection gas pressure is continuously increased, and the resistance value of the microtube electrode is measured in real time until a quasi-stable state is reached, and the injection gas pressure P1 at this time is recorded,

[0006] Step 2: the microtube electrode is completely withdrawn from the cell, so that the intracellular pressure is released through the wound; the injection gas pressure is reduced to the initial value;

[0007] Step 3: the microtube electrode is controlled to puncture into the cell along the same track as the first time, and then retreated to a position where the extrusion shape of the cell is restored; the injection gas pressure is continuously increased, and the resistance value of the microtube electrode is measured in real time until a quasi-stable state is reached, and the injection gas pressure P2 at this time is recorded.

[0008] Step 4: Calculate the difference between P1 and P2 to obtain the intracellular pressure value.

[0009] Further, the quasi-stable state of the electrode solution of the micro-tube electrode: increase the injection gas pressure applied to the micro-tube electrode at a constant speed, and measure the resistance value of the micro-tube electrode, the resistance value decreases with the increase of the injection gas pressure, and the gas pressure-resistance curve is obtained, the difference between the initial stable value and the stable value after the decrease is taken as the transition interval amplitude in the curve, and the resistance value is considered to reach the quasi-stable state when the decrease speed is less than 0.01 times of the transition interval amplitude per second.

[0010] Further, the front ends of the holding micro-tube and the electrode micro-tube are bent to be parallel to the horizontal plane and the axes of the two coincide.

[0011] Further, when measuring the resistance value of the micro-tube electrode, a direct current voltage is applied through the holding micro-tube, and the culture solution where the cell is located is used as the grounding point.

[0012] Further, the micro-tube electrode is made of a glass micro-tube in which a silver wire electrode connected with an amplifier circuit is inserted; the glass micro-tube is installed in front of the needle holder, and the electrode solution is added into the glass micro-tube through the rear injection mode, and the needle holder is connected with the gas circuit to provide the injection gas pressure for the micro-tube electrode.

[0013] Further, the concentration of the electrode solution of the micro-tube electrode: the concentration gap of the electrode solution is increased by 0.1 mol successively until the test experiment is stopped due to the crystallization of the solution with too large concentration to block the pipe opening of the micro-tube electrode; for each concentration of the electrode solution, the injection gas pressure applied to the micro-tube electrode is increased at a constant speed, and the resistance value of the micro-tube electrode is measured, the resistance value decreases with the increase of the injection gas pressure, and the gas pressure-resistance curve is obtained, the ratio of the difference between the initial stable value and the stable value after the decrease to the required injection gas pressure change amount in the 95% change range is taken as an index in the curve, and the solution concentration corresponding to the maximum index value is selected as the concentration of the electrode solution.

[0014] Further, the solution of the micro-tube electrode is KCl solution, and the concentration is 0.8-1.2 mol / L.

[0015] Compared with the prior art, the present application has the beneficial effects that:

[0016] 1. The present application can use ordinary electrode resistance measurement equipment, without the need for micro-force sensors and special equipment with closed-loop gas pressure rapid adjustment function, and therefore is easier to promote than other measurement methods.

[0017] 2. The present application is suitable for high automation, and after the operator moves the holding needle and the injection needle into the field of view, the remaining steps can be automatically completed, and the measurement result is less affected by the professional level of the operator. Attached Figure Description

[0018] Figure 1 This is a flowchart of an embodiment of the present invention;

[0019] Figure 2 This is a circuit model diagram of the microtubular electrode before it penetrates the cell, according to an embodiment of the present invention.

[0020] Figure 3 This is a schematic diagram of the structure of the microtubular electrode after it has been inserted into the cell, according to an embodiment of the present invention.

[0021] Figure 4 This is a circuit model diagram of the microtube electrode after it has penetrated the cell, according to an embodiment of the present invention.

[0022] Figure 5 This is a pressure-resistance curve of KCl solution at different concentrations according to an embodiment of the present invention;

[0023] Figure 6 This is a schematic diagram of a quasi-steady state according to an embodiment of the present invention;

[0024] Figure 7 This is a diagram showing the measurement results of the microtubular electrode penetrating a cell for the first time, according to an embodiment of the present invention.

[0025] Figure 8 This is a diagram showing the measurement results of the microtubular electrode penetrating the cell for the second time, according to an embodiment of the present invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Embodiments of the present invention provide a method for measuring intracellular pressure based on microtubule electrode resistance, such as... Figure 1 As shown, it includes the following steps:

[0028] Preliminary preparations:

[0029] The microtube electrode is drawn from a glass microtube with an inner diameter of 1.17 mm and an outer diameter of 1.5 mm. The opening diameter of the glass microtube tip is approximately 2 micrometers. A silver wire electrode connected to the amplifier circuit is inserted inside the glass microtube. The glass microtube is mounted in front of the needle holder, and electrode solution is added into the glass microtube via a post-injection method. The needle holder is connected to the gas circuit to provide injection pressure for the microtube electrode. When measuring the resistance value of the microtube electrode, both the holding microtube and the microtube electrode are inserted into the culture medium. A DC voltage is applied through the holding microtube, and the culture medium serves as the ground point. The circuit model is as follows. Figure 2 As shown.

[0030] Measurement resistance R of microtube electrode M The resistance R of the silver wire electrodeE The resistance R of the electrode solution inside the microtube L The resistance R caused by the concentration gradient field of the electrode solution and culture medium near the microtube opening I Connected together, that is

[0031] R M =R E +R L +R I ;

[0032] Wherein: the resistance of the silver wire electrode R E R remains constant, but when the volume of liquid entering and exiting the microtube is much smaller than the volume of liquid inside the tube, R... L It can also be considered unchanged.

[0033] R I The concentration gradient field between the corresponding electrode solution and the culture medium is mainly affected by the capillary pressure P between the tube wall and the electrode solution. C and the injection pressure P within the microtube I The effect of P. I When it is small, the capillary pressure P C Cell culture medium is drawn into the microtube, creating a stable concentration gradient field; and when P I As the concentration gradient increases, it will shift towards the microtube opening, causing an increase in the concentration of the electrode solution near the opening, R I Decrease. When P I When increased to a certain extent, the concentration gradient field is completely forced out of the microtube electrode; at this point, R M With P I Further increases will lead to a stable state.

[0034] The above analysis shows that as P I The increase of R M There exists a transition region where the resistance first decreases and then stabilizes. Within this transition region, R... M With pressure P I The changes occur in response to variations in concentration gradient and R. When a microtubule penetrates a cell, the influx of intracellular pressure (intracellular fluid) alters the concentration gradient field. M At this point, you can adjust P. I Re-make R M Reaching a stable state, thus enabling the use of P I Changes are used to measure intracellular pressure.

[0035] When the microtubule electrode pierces the cell, the structure is as follows: Figure 3 As shown, the corresponding circuit model is as follows: Figure 4 As shown. Cell zona pellucida resistance R ZP and cytoplasmic resistance R C Also known as measuring resistance R M Part of, namely:

[0036] R M = R E + R L + R I + R C + R ZP

[0037] wherein: R E , R L , R ZP can be regarded as constants, under the premise that the microtube electrode is sealed with the zona pellucida and the solution volume in and out of the microtube is far less than the cytoplasmic volume in the piercing process, the cytoplasmic resistance R C can be regarded as constant.

[0038] Then, before the intracellular pressure is released after the microtube pierces the cell, R I corresponds to the concentration gradient field of the tube mouth, which is jointly influenced by the intracellular pressure P In , the injection gas pressure P I and the microtube capillary pressure P C ; at this time, by adjusting P I , R M reaches stability again, after which the microtube is withdrawn from the cell to release the intracellular pressure; when the microtube enters the cell again, R I corresponds to the concentration gradient field formed by the injection gas pressure P I and the capillary pressure P C . Therefore, before and after the intracellular pressure is released, the injection gas pressure is adjusted respectively to make the concentration gradient field just be pressed out of the microtube, so that R I reaches stability, according to the formula, that is, R M , the difference between the injection gas pressures when R M is stable can be regarded as the released intracellular pressure value, that is, the intracellular pressure value.

[0039] The sensitivity of R M to the change of the gas pressure is related to the concentration of the microtube electrode solution. Therefore, the KCl concentration of the test electrode solution is increased from 0 mol (pure water) step by step, 0.1 mol each time, until the KCl solution is crystallized and blocked at the tube mouth of the microtube due to too large concentration, and the experiment is stopped; at each concentration, by increasing the gas pressure P I , the resistance value R M is reduced, and R M is stable, the gas pressure-resistance curve is obtained, and the ratio between the difference between the initial R M stable value and the stable value after the resistance is reduced and the required injection gas pressure change amount is taken as the screening index, and the solution concentration corresponding to the maximum index value and the uncrystallized tube mouth is taken as the experimental KCl solution concentration.

[0040] In the specific experiment, it is found that when the KCl concentration is low (0.1 mol / L), with the increase of PI R M The regularity of the change is poor and the transition section is not obvious, as shown in Figure 5 (a). It is not suitable for intracellular pressure measurement; when the KCl solution concentration is 1 mol / L, the transition section (the dotted circle part) of the gas pressure-resistance curve is relatively obvious, which is suitable for intracellular pressure detection, as shown in Figure 5 (b). When the KCl solution density is close to 2 mol / L, as shown in Figure 5 (c), needle mouth crystallization occurs to block the needle tube. Therefore, in this embodiment, the KCl concentration of the microtube electrode solution for intracellular pressure detection is determined to be 1 mol / L.

[0041] Step 1: The cell is fixed by suctioning the microtube. The front ends of the suction microtube and the electrode microtube are bent to be parallel to the horizontal plane and the axes of the two coincide. The microtube electrode is inserted into the cell and then retreated to a position where the extrusion shape of the cell is restored. The injection gas pressure of the microtube electrode is continuously increased at a constant speed, and the resistance value of the microtube electrode is measured in real time until the quasi-steady state is reached, and the injection gas pressure P1 at this time is recorded.

[0042] Quasi-steady state of the electrode solution of the microtube electrode: when the injection gas pressure applied to the microtube electrode is increased at a constant speed and the resistance value is measured, the resistance value decreases with the increase of the injection gas pressure, and the difference between the initial stable value and the stable value after the decrease is taken as the transition interval amplitude in the gas pressure-resistance curve. The resistance value is considered to reach the quasi-steady state when the decrease speed is less than 0.01 times per second, as shown in Figure 6 .

[0043] Step 2: The microtube electrode is completely withdrawn from the cell, so that the intracellular pressure is released through the wound; the injection gas pressure is reduced to the initial value.

[0044] Step 3: The microtube electrode is inserted into the cell along the same trajectory as the first time, and then retreated to a position where the extrusion shape of the cell is restored. The injection gas pressure is continuously increased at a constant speed, and the resistance value of the microtube electrode is measured in real time until the quasi-steady state is reached, and the injection gas pressure P2 at this time is recorded.

[0045] Step 4: Calculate the difference between P1 and P2, which is the intracellular pressure value.

[0046] The method utilizes a general micro-tube electrode resistance measuring device, and automatically judges key states such as micro-needle piercing of the zona pellucida and "quasi-stable state" through an image processing algorithm combined with detection resistance feedback, so as to realize automatic measurement of intracellular pressure. The specific process is as follows: after the micro-tube electrode and the suction micro-tube are moved into the field of view, the system automatically focuses the micro-tube electrode and the suction micro-tube, and after the oocyte is put in, the suction micro-tube automatically completes the suction and fixation of the cell. Then, the injection gas pressure in the micro-tube electrode is increased, so that the electrode resistance value reaches the quasi-stable state. Then, the micro-tube electrode is controlled to penetrate into the cell along the central axis of the suction micro-tube, and the system automatically detects the micro-tube electrode resistance. When the electrode resistance increases by 5 kPa from the quasi-equilibrium state value before penetration, it is considered that the cell has been pierced, and the system controls the micro-tube to withdraw until the cytoplasmic morphology returns to spherical. At this time, the injection gas pressure is increased to make the electrode resistance reach the quasi-stable state, as shown in Figure 7 . Then, the micro-tube electrode withdraws from the cell along the axis of the suction micro-tube, and the micro-tube injection gas pressure is reduced to the value before penetration. After waiting for 30 s, the intracellular pressure is released. Then, the micro-tube electrode enters the cell along the previous track again, and the injection gas pressure is increased again to make the electrode resistance reach the quasi-stable state, as shown in Figure 8 . The intracellular pressure value is determined by the difference between the injection gas pressures before and after the release of the intracellular pressure. The present application measures 20 cells by this method, and the measured intracellular pressure range is 400-600 Pa, which is consistent with the intracellular pressure range of oocytes reported in other literatures, thereby proving the effectiveness of the method of the present application.

[0047] The above describes the present application in detail through examples, but the content described is only exemplary embodiments of the present application, and cannot be considered as limiting the implementation range of the present application. The protection scope of the present application is defined by the claims. Any similar technical solution that utilizes the technical solutions described in the present application, or is inspired by the technical solutions of the present application within the essence and protection scope of the present application, and achieves the above technical effects, or any equivalent changes and improvements to the application scope, shall still belong to the patent coverage protection scope of the present application.

Claims

1. A method for measuring intracellular pressure based on microtube electrode resistance, characterized by, The method comprises the following steps: Step 1: the cell is fixed by the microtubule, the microtubule electrode is inserted into the cell, then the microtubule electrode is retreated to a position where the extrusion shape of the cell is restored, the pressure of the injection gas pressure is continuously increased, and the resistance value of the microtubule electrode is measured in real time until a quasi-stable state is reached, and the pressure P1 of the injection gas pressure at this time is recorded; Step 2: the microtubule electrode is completely withdrawn from the cell, and the intracellular pressure is released through the wound; the pressure of the injection gas pressure is reduced to the initial value; Step 3: the microtubule electrode is inserted into the cell along the same track as the first time, then the microtubule electrode is retreated to a position where the extrusion shape of the cell is restored, the pressure of the injection gas pressure is continuously increased, and the resistance value of the microtubule electrode is measured in real time until a quasi-stable state is reached, and the pressure P2 of the injection gas pressure at this time is recorded; Step 4: the difference between P1 and P2 is calculated to obtain the intracellular pressure value; The quasi-stable state of the electrode solution of the microtubule electrode: the injection gas pressure applied to the microtubule electrode is increased at a constant speed, and the resistance value of the microtubule electrode is measured at the same time; the resistance value decreases with the increase of the injection gas pressure, and the difference between the initial stable value and the stable value after the decrease in the curve is taken as the transition interval amplitude, and the resistance value is considered to reach the quasi-stable state when the decrease speed is less than 0.01 times of the transition interval amplitude per second.

2. The microtubule electrode resistance-based intracellular pressure measurement method according to claim 1, wherein, The front ends of the microtubule and the electrode microtubule are both bent to be parallel to the horizontal plane and the axes of the two coincide.

3. The microtubule electrode resistance-based intracellular pressure measurement method of claim 1, wherein, When the resistance value of the microtubule electrode is measured, a direct current voltage is applied through the microtubule, and the culture solution in which the cell is located is taken as the grounding point.

4. The microtubule electrode resistance-based intracellular pressure measurement method of claim 1, wherein, The microtubule electrode is made of a glass microtubule in which a silver wire electrode connected with an amplifier circuit is inserted; the glass microtubule is installed in front of a needle holder, electrode solution is added into the glass microtubule through the rear injection mode, and the needle holder is connected with a gas circuit to provide the injection gas pressure for the microtubule electrode.

5. The microtubule electrode resistance-based intracellular pressure measurement method of claim 1, wherein, The concentration of the electrode solution of the microtubule electrode: the concentration gap of the electrode solution is increased by 0.1 mol successively until the test experiment is stopped due to the crystallization of the solution with too large concentration to block the pipe opening of the microtubule electrode; for the test experiment of each concentration of the electrode solution, the injection gas pressure applied to the microtubule electrode is increased at a constant speed, and the resistance value of the microtubule electrode is measured at the same time; the resistance value decreases with the increase of the injection gas pressure, and the ratio of the difference between the initial stable value and the stable value after the decrease in the curve to the required injection gas pressure change amount in the 95% change range is taken as an index, and the solution concentration corresponding to the maximum value of the index is selected as the concentration of the electrode solution.

6. The microtubule electrode resistance-based intracellular pressure measurement method of claim 5, wherein, The solution of the microtubule electrode is KCl solution, and the concentration is 0.8-1.2 mol / L.

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