CN111595508B

CN111595508B - A method for measuring intracellular pressure

Info

Publication number: CN111595508B
Application number: CN202010376357.5A
Authority: CN
Inventors: 赵启立; 赵新; 邱金禹; 孙明竹; 孙雨萌
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2021-06-25
Anticipated expiration: 2040-05-07
Also published as: CN111595508A

Abstract

The invention discloses a method for measuring intracellular pressure, comprising the following steps: determining the moment when the cell deformation returns to the highest degree of the initial state during the movement process after the microtubule pierces the cell; The current internal pressure of the cell; calculate the volume change inside the cell before and after the microtubule penetration; calculate the initial intracellular pressure by the volume change ratio before and after the microtubule penetration and the current internal pressure. The invention uses the liquid in the microtubules pierced into the cells to sense the intracellular pressure, detects the cell deformation, the liquid surface contact angle and the liquid surface moving distance in the microtubules through image processing, and then calculates the initial intracellular pressure through the equilibrium pressure model. The invention has simple requirements for experimental equipment, little damage to cells, and does not affect subsequent operations on cells; the initial internal pressure of cells can be effectively detected, and the average speed is 2 minutes per cell.

Description

Method for measuring intracellular pressure

Technical Field

The invention belongs to the technical field of cell-level measurement, and particularly relates to a method for measuring intracellular pressure.

Background

The intracellular pressure is an important influence factor in cell research, and scholars at home and abroad perform a series of preliminary exploration for measuring the intracellular pressure, and specific measuring methods comprise a magnetic tweezers method and an oil injection method. The magnetic tweezers method utilizes small magnetic beads driven by a magnetic field to extrude cells before and after membrane rupture, researches the change of a stress-deformation curve of the cells, and measures the internal pressure of the cells. The method needs to install a laser beam emitter, control magnetic poles and other equipment, has high requirements on hardware, and can influence the subsequent operation of the system due to an excessively complex system. The operation difficulty of releasing the internal pressure and controlling the magnetic beads is high, the steps are complex, the response speed is slow, and the capacity of measuring the internal pressure of cells in real time is not realized. The oil injection method firstly carries out off-line calibration on the relation between the intracellular pressure and the oil injection amount, then injects oil drops into cells, and detects the intracellular pressure according to the volume of the measured oil drops. The method has low requirement on equipment and can realize real-time detection, but in order to measure the volume of the injection liquid, an immiscible oil body ball needs to be formed in the cell, the injection success rate is low, the activity of the cell can be damaged, the measured cell is dead, and the subsequent operation cannot be carried out. Therefore, it is necessary to find a method for measuring intracellular pressure with simple operation and little damage to cells.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a method for measuring the intracellular pressure, which comprises the steps of sensing the intracellular pressure by using a microtubule which is penetrated into a cell, detecting the cell deformation and the contact angle and the position of the liquid level in the microtubule by using image processing, and then calculating the initial intracellular pressure of the cell by using a balanced pressure model to realize the measurement of the intracellular pressure.

The technical scheme adopted by the invention is as follows: a method for measuring intracellular pressure, comprising the steps of:

a. and automatically detecting the cell deformation, determining the time when the cell deformation is recovered to the highest degree in the movement process after the microtubules penetrate into the cells, and approximately considering that the cell volume at the time is equal to the initial cell volume.

The method for measuring cell deformation comprises the steps of firstly identifying undeformed cells through Hough circle detection, detecting the contour of cytoplasm of the cells, then carrying out binarization processing on a cell image, carrying out expansion corrosion processing on the binarized image, detecting the outer contour of a cell zona pellucida through edge detection, then tracking the displacement of each part of the cells in the process of microtubule penetration by adopting an optical flow method, carrying out edge detection on the region where the cells are located, calculating the number of pixels occupied by the cells to judge whether the deformation is recovered to the initial state, detecting a frame with the best cell deformation recovery in the process of microtubule penetration and withdrawal, and approximately considering that the cell deformation is recovered to the initial state at the moment.

b. And automatically detecting the contact angle of the liquid level in the microtube, detecting the capillary pressure of the liquid in the microtube through a liquid equilibrium pressure model in the microtube and a Poplar equation, and calculating the current pressure inside the cell by combining the injection air pressure detected by an air pressure sensor.

The current method for detecting the pressure inside the cell is the detection of the contact angle of the liquid level in the microtube, when the microtube with the extremely small size is immersed in the culture solution, liquid molecules in the microtube can be pulled backwards by the attraction force of the tube wall for the tube wall of the hydrophilic glass microtube, and meanwhile, under the action of the liquid surface tension, the liquid molecules near the liquid level can generate a pulling force trying to recover the liquid level, namely capillary force F_c. Under these two forces, the gas-liquid interface within the microtube will appear as a concave surface. The concave surface can generate additional pressure on the liquid, so that the pressure inside and outside the liquid surface is unequal, the pressure outside the liquid surface is constantly higher than the pressure inside the liquid, and the phenomenon is called capillary phenomenon. And (3) detecting the capillary pressure of the liquid in the microtubule through a liquid equilibrium pressure model in the microtubule and the Poplar equation, and calculating the current pressure inside the cell by combining the injection pressure detected by the air pressure sensor. The capillary force generated by the liquid surface is given by the following equation:

F_c＝σ2πRcosβ

wherein sigma is the surface tension coefficient of the cell culture solution, which can be measured by a drawing method, beta represents the contact angle between the liquid level and the tube wall, the liquid level is partially binarized, after the edge is detected, curve fitting is carried out, the derivative of the liquid level and the tube wall is obtained, and the cosine value of the contact angle is calculated. The contact angle of the liquid surface depends on the hydrophilicity of the inner wall material of the microtube and the surface tension coefficient of the culture solution, and the influence of pressure.

The equilibrium pressure model is given by the following equation:

F_inj＝F_in+F_m+F_c；

F_injthe force of the injection air pressure on the liquid in the tube is expressed by the following formula:

F_inj＝πR²P_inj；

wherein R represents the inner diameter of the microtube at the liquid level; p_injIndicating the injection air pressure.

F_inIndicating intracellular pressure generating in-tube liquidIs obtained by the following formula:

F_in＝πR₀ ²P_in；

wherein R is₀Denotes the inner diameter of the micro-orifice, P_inIndicating the intracellular pressure.

F_mThe force of the microtube wall against the liquid is expressed by the following formula:

F_m＝π(R²-R₀ ²)P_in；

F_cthe capillary force, which is a component force in the horizontal direction, is expressed by the following equation:

F_c＝σ2πRcosβ；

wherein sigma is the surface tension coefficient of the cell culture solution, and can be measured by a drawing method, and beta is simultaneously determined by the hydrophilicity of the inner wall material of the microtube and the surface tension coefficient of the culture solution.

Under the condition that the liquid level is static, the liquid in the micro-tube is stressed in a balanced manner, and under the condition that the friction force between the liquid and the inner wall of the micro-tube is neglected, the detected pressure intensity at the current moment inside the cell can be finally given by the following formula according to a balanced pressure model:

P_in＝P_inj-(2σcosβ)/R。

c. and automatically detecting the moving distance of the liquid level in the microtube, wherein the interior of the cell is communicated with the microtube liquid after the microtube penetrates into the cell, and the liquid level in the microtube moves due to the existence of the internal pressure of the cell.

The method for detecting the liquid level moving distance is to respectively detect the real-time positions of the liquid level and the microtube through template matching, and the difference change of the positions is the liquid level moving distance delta L.

d. The initial intracellular pressure was calculated from the ratio of the volume change of the inside of the cell before and after the penetration of the microtube and the current pressure inside the cell.

The current intracellular pressure value is related to the initial intracellular pressure by:

wherein P is_originRepresenting the initial intracellular pressure of the cell, P' representing the current intracellular pressure detected in step b, V_cellIndicating the cell volume prior to penetration. In conjunction with the equilibrium pressure model, the final measured intracellular pressure can be given by the following equation:

compared with the prior art, the invention has the beneficial effects that:

1. the method comprises the steps of sensing the intracellular pressure by utilizing liquid in a microtubule which penetrates into a cell, detecting the deformation of the cell, the contact angle of the liquid level in the microtubule and the moving distance of the liquid level through image processing, and then calculating the initial intracellular pressure through a balanced pressure model;

2. the experimental equipment of the invention has simple requirement, small damage to cells and no influence on the subsequent operation on the cells;

3. the present invention can effectively detect the initial internal pressure of the cells, and the average speed is 2 minutes per cell.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a graph showing the detection of cell deformation according to the present invention;

FIG. 3 is a schematic view of the liquid level in the microtube of the present invention under force;

FIG. 4 is a graph of a curve fit of the detected tube wall to liquid level of the present invention;

FIG. 5 is a schematic view of the liquid in the microtube of the present invention under force;

FIG. 6 is a matching graph of a template for selecting an attachment immobilized on a microtube according to the present invention;

FIG. 7 is a view showing the movement of the liquid level and the microtube according to the present invention;

fig. 8 is a liquid surface moving distance diagram of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The embodiment of the invention discloses a method for measuring intracellular pressure, and the flow is shown in figure 1.

(1) Domestic pig oocyte retrieval

The oocytes used in this example were from pig oocytes taken from a local slaughterhouse, and after removal of the pig ovaries from the slaughterhouse, the oocytes were transported to the laboratory within two hours using a thermos bottle filled with 35 ° to 37 ° physiological saline. Then immediately washed twice with 37 ℃ sterile saline containing 100IU/L penicillin and 50mg/L streptomycin. Oocytes were aspirated from follicles of 2-6mm diameter on the ovary, and after the aspirated cells were washed three times with TL-Hepes-PVA, in vitro maturation culture (IVM) was performed in an incubator at 39 ℃ and 5% carbon dioxide concentration for 42 hours. After IVM, cells were deoccluded with 0.1% hyaluronidase. Finally, the cells were washed three times with M199 to obtain the oocytes used.

(2) The cell deformation test is shown in figure 2:

in this example, the inner radius of the microtube is about 10 μm, the cell radius is about 75-80 μm, and the image resolution is 450X 520 pixels. Fig. 2(a) shows deformation of zona pellucida and cytoplasm tracked during deformation after cell insertion, and fig. 2(b) shows the best frame for recovery of cell deformation detected during needle withdrawal, i.e. the frame with the number of pixels covered by the cell closest to the initial state. FIG. 2(c) is a graph showing the change in the number of pixel points covered by the cells in the whole process.

(3) The contact angle of the liquid surface in the microtube was measured as shown in fig. 3 and 4:

when a microtube with an extremely small size is immersed in a culture solution, liquid molecules in the microtube are pulled backwards by the attraction of the wall of the hydrophilic glass microtube and at the same time, the liquid molecules near the liquid surface generate a pulling force trying to restore the liquid surface under the action of the surface tension of the liquid, namely capillary force F_c. Under the action of these two forces, the force,the gas-liquid interface within the microtube will appear as a concave surface. The concave surface can generate additional pressure on the liquid, so that the pressure inside and outside the liquid surface is unequal, and the pressure outside the liquid surface is constantly greater than the pressure inside the liquid, namely the capillary pressure. FIG. 3 shows the force profile of the liquid surface, where F_NIndicating that the liquid molecule is pulled by the wall of the tube, F_SIndicating the pulling force that restores the fluid level.

After the liquid level is partially binarized and the edge is detected, curve fitting is performed to obtain the derivative of the liquid level and the pipe wall, and the cosine value of the contact angle is calculated. FIG. 4 shows the results of a curve fit of the detected wall to the liquid level, the capillary force being obtained from the following equation:

F_c＝σ2πRcosβ

fig. 5 shows the stress condition of the liquid in the microtube, the liquid in the microtube is subjected to the balance of four forces, namely the force generated by the injection air pressure on the liquid in the microtube, the pressure generated by the intracellular pressure on the liquid in the microtube, the force of the microtube wall on the liquid, and the component force in the horizontal direction, namely the capillary force, to obtain a balanced pressure model:

F_inj＝F_in+F_m+F_c

wherein F_injIndicating the force of the injection gas pressure on the liquid in the tube, F_inShowing the pressure of the intracellular pressure on the liquid in the tube, F_mIndicating the force of the microtube walls against the liquid.

(4) Detecting the moving distance of the liquid level in the microtube in one frame of deformation recovery, as shown in fig. 6, 7 and 8:

in order to detect the movement of the microtube, the embodiment selects the attachments fixed on the microtube for tracking, selects the template for fixing the attachments on the microtube and the liquid level template through a template matching method, and performs template matching on each frame in the movement process to obtain the movement condition of the liquid level and the microtube. The change of the distance between the microtube and the liquid level is the movement of the liquid level in the puncturing process. Fig. 6 shows the selected template for matching, and fig. 7 shows the detected liquid level and the movement of the microtube. In order to obtain a more accurate liquid level moving distance, the distance change is sub-pixel processed, and the result is shown in fig. 8.

(5) The initial intracellular pressure was calculated from the ratio of the volume change of the inside of the cell before and after the penetration of the microtubules:

the puncture experiment of 20 oocytes is completed within 40 minutes, and required parameters of injection pressure, deformation recovery frames, contact angles between liquid level and tube wall, liquid level moving distance, cell inner diameter and microtubule inner diameter are detected in sequence to calculate the intracellular pressure.

The present invention has been described in detail with reference to the embodiments, but the description is only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The scope of the invention is defined by the claims. The technical solutions of the present invention or those skilled in the art, based on the teaching of the technical solutions of the present invention, should be considered to be within the scope of the present invention, and all equivalent changes and modifications made within the scope of the present invention or equivalent technical solutions designed to achieve the above technical effects are also within the scope of the present invention.

Claims

1. a measuring method of intracellular pressure, is characterized in that: comprise the following steps:

a. Determine the moment when the cell deformation returns to the highest degree of the initial state during the movement process after the microtubule penetrates the cell;

b. Calculate the current internal pressure of the cell according to the capillary pressure of the liquid in the microtube and the injection air pressure;

c. Calculate the volume change inside the cell before and after microtubule penetration;

d. Calculate the initial intracellular pressure by the volume change ratio inside the cell before and after microtubule piercing and the current pressure inside the cell,

The relationship between the current intracellular pressure and the initial intracellular pressure is:

where P _origin represents the initial internal pressure of the cell, P′ represents the detected current intracellular pressure, V _cell represents the cell volume before puncture, R represents the inner diameter of the microtubule liquid surface, and ΔL represents the distance the liquid level moves.

2. The method for measuring intracellular pressure according to claim 1, wherein in step a, the method for detecting cell deformation is to detect undeformed cells by Hough circle detection, and then use optical flow for the cell part The deformation process is tracked by the method, edge detection is performed on the area where the cell is located, and the number of pixels occupied by the cell is calculated to judge whether the deformation returns to the initial state.

3. the measuring method of intracellular pressure as claimed in claim 1 or 2, is characterized in that: in step b, the capillary pressure of liquid in micropipe is detected by liquid equilibrium pressure model in micropipe and Young Laplace equation, The injection pressure is detected by the air pressure sensor.

4. The method for measuring intracellular pressure as claimed in claim 3, characterized in that: the equilibrium pressure model comprises the force produced by the injection air pressure on the liquid in the tube, the pressure produced by the intracellular pressure on the liquid in the tube, and the pressure of the microtube wall on the liquid. force and component forces in the horizontal direction.

5. The method for measuring intracellular pressure according to claim 1 or 2, characterized in that: in step c, at the moment when the degree of cell deformation recovery is the highest, the amount of change in the internal volume of the cell can be regarded as the material inside the cell entering the microscopic cell. The amount of the tube can be calculated from the distance the liquid surface moves in the microtube.

6. The measuring method of intracellular pressure as claimed in claim 5, is characterized in that: the method for detecting liquid level moving distance is to detect the real-time position of liquid level and microtubule respectively by template matching, and the difference of its position changes namely is the liquid level moving distance.