CN115128303A - A method for measuring Young's modulus of single cells based on atomic force nanoindentation - Google Patents

Abstract

The invention discloses a method for measuring Young modulus of a single cell based on atomic force nano indentation, and belongs to the technical field of nano measurement. Respectively arranging indentation points at the central position and the edge position of the cell by using an atomic force microscope to obtain an indentation curve, reading indentation data, analyzing the indentation curve by using a Hertz model to obtain the Young modulus of each indentation depth, and obtaining the distribution condition of the cell skeleton in the cell by researching and analyzing the elastic characteristics of the central position and the edge position of the cell. The invention discloses a new method for determining an initial calculation point, which eliminates the interference of a substrate on the elastic modulus of cells and obtains more accurate Young modulus and cytoskeleton distribution.

Description

A method for measuring Young's modulus of single cells based on atomic force nanoindentation

technical field

The invention belongs to the technical field of nanometer measurement, in particular to a method for measuring Young's modulus of a single cell based on atomic force nanoindentation.

Background technique

Since the invention of the atomic force microscope, it has been widely used in the field of biomechanical engineering, especially in the detection of the mechanical properties of living cells, due to its simple sample preparation and nanoscale resolution. The biomechanical properties of cells, such as cell elasticity, viscosity and stiffness, are important essential characteristics of cells, and are closely related to the pathological changes and life activities of living organisms. On the one hand, changes in cell mechanical properties will directly affect physiological functions and cause diseases; on the other hand, the occurrence of diseases can also lead to changes in cell structure and mechanical properties. The mechanical properties of cells affect the ability of cells to deform, move, and feel external stimuli in the microenvironment. Studies have shown that abnormal cell elasticity is related to the occurrence and development of diseases. The Young's modulus is used to quantify cell elasticity, according to the Young's modulus. The changes in the diseased cells distinguish the diseased cells from the normal cells, making it a biomarker, providing a new idea for the early diagnosis and treatment of cancer.

The Hertz model is currently the most classic model for analyzing the force-displacement curve of AFM. At present, there are two main methods for calculating cell elasticity using the Hertzian model: the two-point method and the slope method. In 2014, Wang Zhe et al. conducted a comparative study on the AFM indentation curve analysis methods. By comparing two AFM force-displacement curve analysis methods based on the Hertz model, they found that the elastic modulus of cells increased with the increase of indentation depth. The research shows that the change trend of the slope method curve is gentle, indicating that the slope method can reduce the calculation error of Young's modulus caused by the wrong judgment of the contact point (see Wang Zhe, Hao Fengtao, Chen Xiaohu, Yang Zhouqi, Ding Chong, Shang Peng. Atomic force microscope pressure Comparative study of trace curve analysis methods[J].Journal of Biomedical Engineering,2014,31(05):1075-1079). Although the slope method does not need to find the contact point, the slope can be directly obtained by linearizing the power of the original data, but the indentation depth cannot be accurately obtained, and the calculation process is complicated. Although the calculation of the two-point method is simple, the determination of the contact point is affected by many factors, resulting in an increase in the calculation error of Young's modulus. The main influencing factor is the determination of the initial calculation point. The inaccuracy of the initial calculation point may cause cell An over- or underestimation of elasticity. Compared with the existing calculation method, the method for calculating Young's modulus introduced by the present invention obtains two indentation depths on the cell, and takes the smaller indentation depth as the starting calculation point to obtain the Young's modulus determined by the cytoskeleton. Modulus, the calculation process is simple and the result is accurate.

SUMMARY OF THE INVENTION

The technology of the present invention solves the problem: overcomes the deficiencies of the prior art, provides a method for measuring the Young's modulus of a single cell based on atomic force nano-indentation, and solves the problem that the two-point method of the Hertz model can not determine the initial calculation point and cause Young's Due to the large error of modulus calculation, this method can simultaneously obtain the accurate indentation depth to determine the influence of the cytoskeleton on the Young's modulus of the cell and the distribution of the cytoskeleton inside the cell.

The technical solution adopted by the present invention to solve the technical problem is as follows: (1) first obtain a set of indentation curves on a blank substrate, and then set indentation points in a 3×3 array at the cell center position and edge position respectively; (2) Set different forces on the same indentation point to obtain different indentation depths; (3) Use the cross-correlation algorithm to find the data segment with higher correlation coefficient between the cell force-displacement curve and the base force-displacement curve, and delete this segment of data , and compensate it, and then extend the remaining data segment to the original length to obtain a corrected force-displacement curve corrected by the cross-correlation algorithm; (4) Using the two-point method of the Hertz contact model, a small force The obtained indentation depth was used as the initial calculation point, and the cell force-displacement curve and the corrected force-displacement curve were analyzed and calculated to obtain a graph of Young's modulus varying with the indentation depth.

After the present invention adopts the above-mentioned technical scheme, it mainly has the following advantages:

(1) Compared with the traditional method, the method designed by the present invention provides a new method for determining the initial calculation point, which avoids that the value of Young's modulus is too large due to the inaccurate determination of the initial calculation point. or too small;

(2) The present invention sets the indentation points at the center position and the edge position of the cell, and determines the distribution of the cytoskeleton at the center position and the edge position of the cell by analyzing the difference of the Young's modulus of the cell;

(3) The present invention finds the part with a larger correlation coefficient between the cell force-displacement curve and the base force-displacement curve through the cross-correlation algorithm, and determines it as the influence of the base on the measurement result, deletes this section of data, and removes the base on the measurement. impact on results.

Description of drawings

Fig. 1 is a flowchart of a method for measuring Young's modulus of a single cell based on atomic force nanoindentation proposed by the present invention;

2 is a schematic diagram of the cell indentation curve obtained by the atomic force microscope of the present invention;

3 is a schematic diagram of obtaining a modified force-displacement curve of the Young's modulus calculation method based on atomic force nanoindentation proposed by the present invention;

Fig. 4 is the indentation depth-Young's modulus curve obtained by analyzing the force-displacement curve of the present invention, (a) the indentation depth-Young's modulus curve obtained by analyzing the cell force-displacement curve, (b) analyzing the correction force - Indentation depth-Young's modulus curve obtained from the displacement curve;

Fig. 5 is the indentation depth-Young's modulus curve obtained at the center position and the edge position of the cell according to the present invention.

Detailed ways

The examples of the present invention will be described in further detail below with reference to the accompanying drawings.

As shown in FIG. 1, it is a flow chart of measuring the Young's modulus of a single cell based on atomic force nanoindentation according to the present invention. A method for measuring the Young's modulus of a single cell based on atomic force nanoindentation mainly includes: (1) firstly in Obtain a set of indentation curves on a blank substrate, and then set indentation points in a 3×3 array at the center and edge positions of the cells respectively; (2) Set different indentations on the same indentation point to obtain different indentations (3) Use the cross-correlation algorithm to find the data segment with higher correlation coefficient between the cell force-displacement curve and the base force-displacement curve, delete this segment of data, compensate for it, and then extend the remaining data segment to the original length , to obtain a corrected force-displacement curve corrected by the cross-correlation algorithm; (4) Using the two-point method of the Hertzian contact model, the cell force-displacement curve was analyzed and calculated by taking the indentation depth obtained by the small force as the initial calculation point. and the corrected force-displacement curve to obtain a graph of Young's modulus as a function of indentation depth.

As shown in Figure 2, it is the principle diagram of the cell indentation curve obtained by the atomic force microscope in the contact mode, a is that the probe has not yet touched the cell, and the force-displacement curve is parallel to the coordinate axis; b is that the probe begins to touch the cell In the cell, the force-displacement curve begins to deflect; c is the probe deep into the cell, and the deflection of the force-displacement curve increases; d is the indentation point set on the cell.

As shown in Figure 3, it is a schematic diagram of the acquisition of the modified force-displacement curve of the present invention. Figure a is a force-displacement curve intercepted by taking the indentation depth obtained by a small force as a contact point, and Figure b is a cross-correlation algorithm. The force-displacement curve of the data segment with a higher correlation coefficient between the force-displacement curve and the base force-displacement curve is deleted. Figure c shows the corrected force-displacement curve extending the remaining data segment to the original length.

As shown in FIG. 4 , it is a graph of the indentation depth-Young's modulus obtained by using the Hertzian contact model to analyze the cell force-displacement curve and the corrected force-displacement curve. As shown in Figure a, the calculated indentation depth-Young's modulus curve is analyzed using the cell force-displacement curve. Due to the influence of the substrate, the tail of the indentation depth-Young's modulus curve is raised, indicating that the substrate will It has an impact on the measurement of Young's modulus of cells; as shown in Figure b, the calculated indentation depth-Young's modulus curve is analyzed by using the corrected force-displacement curve. Due to the effect of the cross-correlation algorithm, the influence of the substrate is eliminated, and finally the Young's modulus value tends to be stable.

As shown in FIG. 5 , it is a graph of the indentation depth-Young's modulus of the present invention at the center position and the edge position of the cell. Figure a is the indentation depth-Young's modulus curve obtained by analyzing the corrected force-displacement curve at the center of the cell, and Figure b is the indentation depth-Young's modulus curve obtained by analyzing the corrected force-displacement curve at the edge of the cell .

Example 1:

Figure 4 shows the indentation depth-Young's modulus curve before and after the correction according to the method of the present invention obtained according to the flow chart of Figure 1. The cells were seeded on a cover glass and placed in an incubator. After 24 hours, the Rinse the coverslip with PBS, remove the floating cells on the coverslip, place the rinsed coverslip in a petri dish, add clean culture medium, and place the petri dish with coverslips on the AFM sample stage . Cells were imaged in contact mode, then indentation points were set on the cells in force spectroscopy mode, and indentation curves were obtained using two applied forces of different magnitudes on the same indentation point. Use the data analysis software to read the indentation curve data, take the indentation depth obtained by the small force as the starting point for calculation, and use the cross-correlation algorithm to eliminate the influence of the substrate on the calculation result of Young's modulus, and obtain the elasticity determined by the cytoskeleton. feature.

Example 2:

Figure 5 shows the indentation depth-Young's modulus curve obtained according to the flow chart of Figure 1 before and after the correction by the method of the present invention. The cells were seeded on a cover glass and placed in an incubator. After 24 hours, the Rinse the coverslip with PBS, remove the floating cells on the coverslip, place the rinsed coverslip in a petri dish, add clean culture medium, and place the petri dish with coverslips on the AFM sample stage ; Imaging the cells in contact mode, then set indentation points at the center and edge of the cells respectively, and apply two different forces on the same indentation point to obtain the indentation curve; use data analysis software to read Take the indentation curve data, take the indentation depth obtained by the small force as the starting point for calculation, use the cross-correlation algorithm to eliminate the influence of the substrate on the calculation result of Young's modulus, and obtain the indentation depth at the center and edge of the cell - Young The elastic characteristics determined by the cytoskeleton are obtained by the modulus curve, and the distribution of the cytoskeleton in the center position and the edge position is obtained.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (4)

1. A method for measuring the Young modulus of a single cell based on atomic force nano indentation is characterized by comprising the following steps: performing nano indentation and measuring the elasticity of cells in an atomic force microscope contact mode, and comprising the following steps of: (1) firstly, acquiring a group of indentation curves on a blank substrate, and then respectively arranging indentation points in a 3 x 3 array form at the center position and the edge position of a cell; (2) setting different acting forces on the same indentation point to obtain different indentation depths; (3) finding a data segment with higher correlation coefficient between the cell force-displacement curve and the substrate force-displacement curve by using a cross-correlation algorithm, deleting the data segment, compensating the data segment, and extending the rest data segment to the original length to obtain a corrected force-displacement curve corrected by the cross-correlation algorithm; (4) and analyzing and calculating a cell force-displacement curve and a correction force-displacement curve by using a two-point method of a Hertz contact model and taking the indentation depth obtained by small acting force as an initial calculation point to obtain a curve graph of Young modulus changing along with the indentation depth.

2. The method according to claim 1, wherein the step (1) sets indentation points at the center and edge positions of the cell, wherein: the distribution of intracellular cytoskeleton is determined by studying the difference of the Young modulus of the cells at different positions.

3. The method of claim 1, wherein step (2) provides different magnitudes of force at the same indentation point, wherein: the effect of the substrate on the measurement of the young's modulus of the cells was determined by studying the indentation depth of the cells by the effect of the different forces.

4. The method according to claim 1, wherein the step (3) uses a cross-correlation algorithm to find the data segment with higher correlation coefficient between the cell force-displacement curve and the substrate force-displacement curve, and is characterized in that: and determining the Young modulus of the cells independent of the substrate by using a cross-correlation algorithm, and eliminating the influence of the substrate.

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