CN115128303B - Method for measuring Young modulus of single cell based on atomic force nanoindentation - Google Patents
Method for measuring Young modulus of single cell based on atomic force nanoindentation Download PDFInfo
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- CN115128303B CN115128303B CN202110311363.7A CN202110311363A CN115128303B CN 115128303 B CN115128303 B CN 115128303B CN 202110311363 A CN202110311363 A CN 202110311363A CN 115128303 B CN115128303 B CN 115128303B
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- 238000000034 method Methods 0.000 title claims abstract description 35
- 210000004027 cell Anatomy 0.000 claims abstract description 73
- 238000007373 indentation Methods 0.000 claims abstract description 64
- 238000004364 calculation method Methods 0.000 claims abstract description 24
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 210000004292 cytoskeleton Anatomy 0.000 claims abstract description 8
- 238000006073 displacement reaction Methods 0.000 claims description 40
- 238000012937 correction Methods 0.000 claims description 6
- 238000005259 measurement Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 3
- 230000002035 prolonged effect Effects 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims 1
- 239000000523 sample Substances 0.000 description 7
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- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
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- G01Q30/20—Sample handling devices or methods
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract
The invention discloses a method for measuring Young modulus of a single cell based on atomic force nanoindentation, and belongs to the technical field of nanomeasurement. An atomic force microscope is used for respectively setting indentation points at the central position and the edge position of the cell to obtain an indentation curve, indentation data are read, the indentation curve is analyzed by using a Hertz model to obtain Young modulus of each indentation depth, and the distribution condition of a cytoskeleton in the cell is obtained by researching and analyzing the elastic characteristics of the central position and the edge position of the cell. The invention discloses a novel 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
Technical Field
The invention belongs to the technical field of nano measurement, and particularly relates to a method for measuring Young modulus of a single cell based on atomic force nano indentation.
Background
Since atomic force microscope invention, the sample preparation is simple, and the sample has the advantages of nano-scale resolution, and the like, and is widely applied to the field of biomechanical engineering, in particular to the detection and research of the mechanical properties of living cells. The biomechanical properties of cells, such as elasticity, viscosity and rigidity of cells, are important essential characteristics of cells, and are closely related to pathological changes and vital activities of living bodies. On the one hand, the change of the mechanical property of the cells can directly influence the physiological function, and cause the occurrence of diseases; on the other hand, the occurrence of diseases can also lead to changes in cellular structure and mechanical properties. The mechanical properties of the cells influence the deformation and movement of the cells and the ability of the cells to feel external stimulus in a microenvironment, and researches show that the abnormal cell elasticity is related to the occurrence and development of diseases, the cell elasticity is quantified through the Young modulus, and diseased cells are distinguished from normal cells according to the change of the Young modulus, so that the diseased cells are a biomarker, and a new idea is provided for early diagnosis and treatment of cancers.
The hertz model is the most classical model for analyzing the force-displacement curve of an atomic force microscope at present. The current methods for calculating cell elasticity by using the Hertz model mainly comprise a two-point method and a slope method. In 2014 Wang Zhe et al, a comparative study was conducted on an atomic force microscope indentation curve analysis method, and by comparing two atomic force microscope force-displacement curve analysis methods based on a Hertz model, it was found that the elastic modulus of cells decreases with increasing indentation depth, and the study showed that the trend of the slope curve was gentle, indicating that the slope method can reduce Young's modulus calculation errors due to erroneous judgment of contact points (see Wang Zhe, hao Fengtao, chen Xiaohu, yang Zhouqi, ding Chong, comparative study [ J ] of atomic force microscope indentation curve analysis method, journal of biomedical engineering, 2014,31 (05): 1075-1079). The slope method can directly linearize the original data to obtain the slope by multiplying without searching for the contact point, but the indentation depth cannot be accurately obtained, and the calculation process is complex. Although the two-point method calculation is simple, the judgment of the contact point is influenced by a plurality of factors, so that the Young modulus calculation error is increased, wherein the main influencing factor is the judgment of the initial calculation point, and the inaccuracy of the initial calculation point can cause the overestimation or the underestimation of the cell elasticity. Compared with the existing calculation method, the method for calculating the Young modulus provided by the invention has the advantages that two indentation depths are obtained on cells, the smaller indentation depths are used as initial calculation points to obtain the Young modulus determined by the cytoskeleton, the calculation process is simple, and the result is accurate.
Disclosure of Invention
The technical solution of the invention is as follows: the method solves the problem of large Young modulus calculation error caused by incapability of judging an initial calculation point by a two-point method of a Hertz model, and can simultaneously obtain accurate indentation depth to determine the influence of cytoskeleton on the Young modulus of cells and the distribution of the cytoskeleton in the cells.
The technical scheme adopted for solving the technical problems is as follows: (1) Firstly, a group of indentation curves are obtained on a blank substrate, and indentation points are respectively arranged at the center position and the edge position of the cell in a 3X 3 array mode; (2) Setting different acting forces on the same indentation point to obtain different indentation depths; (3) The data segment with higher correlation coefficient between the cell force-displacement curve and the basal force-displacement curve is found by using a cross-correlation algorithm, the data segment is deleted, the data segment is compensated, and the rest data segment is prolonged to the original length, so that a corrected force-displacement curve corrected by the cross-correlation algorithm is obtained; (4) And using a two-point method of the Hertz contact model, taking the indentation depth obtained by small acting force as an initial calculation point, analyzing and calculating a cell force-displacement curve and a correction force displacement curve, and obtaining a graph of Young modulus changing along with the indentation depth.
After the technical scheme is adopted, the invention has the following advantages:
(1) Compared with the traditional method, the method provided by the invention provides a new method for determining the initial calculation point, and the situation that the Young modulus value is too large or too small due to inaccurate determination of the initial calculation point is avoided;
(2) The invention sets indentation points at the center position and the edge position of the cell, and judges the distribution of the cytoskeleton at the center position and the edge position of the cell by analyzing the Young modulus difference of the cell;
(3) The invention finds out the part with larger correlation coefficient of the cell force-displacement curve and the basal force-displacement curve through the cross-correlation algorithm, judges the part as the influence of the basal on the measurement result, deletes the data, and removes the influence of the basal on the measurement result.
Drawings
FIG. 1 is a flow chart of a method for measuring Young's modulus of single cells based on atomic force nanoindentation according to the present invention;
FIG. 2 is a schematic diagram of an atomic force microscope of the present invention for obtaining a cell indentation curve;
FIG. 3 is a schematic diagram showing the acquisition of a correction force-displacement curve based on the Young's modulus calculation method of atomic force nanoindentation according to the present invention;
FIG. 4 is an indentation depth-Young's modulus curve obtained by analyzing a force-displacement curve, (a) an indentation depth-Young's modulus curve obtained by analyzing a cell force-displacement curve, and (b) an indentation depth-Young's modulus curve obtained by analyzing a corrected force-displacement curve according to the present invention;
FIG. 5 is a graph showing the indentation depth versus Young's modulus obtained at the center and edge positions of a cell according to the present invention.
Detailed Description
Examples of the present invention will be described in further detail below with reference to the accompanying drawings.
As shown in fig. 1, in the flow chart of measuring young's modulus of single cells based on atomic force nanoindentation of the present invention, a method for measuring young's modulus of single cells based on atomic force nanoindentation mainly comprises: (1) Firstly, a group of indentation curves are obtained on a blank substrate, and indentation points are respectively arranged at the center position and the edge position of the cell in a 3X 3 array mode; (2) Setting different acting forces on the same indentation point to obtain different indentation depths; (3) The data segment with higher correlation coefficient between the cell force-displacement curve and the basal force-displacement curve is found by using a cross-correlation algorithm, the data segment is deleted, the data segment is compensated, and the rest data segment is prolonged to the original length, so that a corrected force-displacement curve corrected by the cross-correlation algorithm is obtained; (4) And using a two-point method of the Hertz contact model, taking the indentation depth obtained by small acting force as an initial calculation point, analyzing and calculating a cell force-displacement curve and a correction force displacement curve, and obtaining a graph of Young modulus changing along with the indentation depth.
As shown in fig. 2, a is a schematic diagram of an atomic force microscope for obtaining an indentation curve of a cell in a contact mode, and a is that a probe is not contacted with the cell yet, and a force-displacement curve is parallel to a coordinate axis; b is that the probe starts to contact with the cell, and the force-displacement curve starts to deflect; c is that the probe penetrates into the cell, so that the deflection degree of the force-displacement curve is increased; d is the indentation point set on the cell.
Fig. 3 is a schematic diagram showing the acquisition of a corrected force-displacement curve according to the present invention, wherein fig. a is a force-displacement curve taken by taking the depth of an indentation obtained by a small force as a contact point, fig. b is a force-displacement curve obtained by deleting a data segment with a higher correlation coefficient between a cellular force-displacement curve and a basal force-displacement curve by using a cross-correlation algorithm, and fig. c is a corrected force-displacement curve obtained by extending the remaining data segment to an original length.
As shown in FIG. 4, an indentation depth-Young's modulus graph obtained by analyzing a cell force-displacement curve and correcting the force-displacement curve by using a Hertz contact model is shown in the present invention. As shown in fig. a, the indentation depth-young modulus curve obtained by calculation is analyzed by using a cell force-displacement curve, and the tail part of the indentation depth-young modulus curve is tilted due to the influence of the substrate, which indicates that the substrate can have an influence on the young modulus measurement of cells; as shown in fig. b, the indentation depth-young's modulus curve obtained by calculation is analyzed by using the corrected force-displacement curve, the influence of the substrate is eliminated due to the effect of the cross-correlation algorithm, and finally the young's modulus value is stabilized.
FIG. 5 is a graph showing the indentation depth-Young's modulus curve of the present invention at the center and edge positions of the cell. Fig. a is an indentation depth-young's modulus curve obtained by analyzing a corrected force-displacement curve at a cell center position, and fig. b is an indentation depth-young's modulus curve obtained by analyzing a corrected force-displacement curve at a cell edge position.
Example 1:
FIG. 4 shows the indentation depth-Young's modulus curve before and after correction according to the method of the present invention, which is obtained according to the flowchart of FIG. 1, cells were inoculated onto a coverslip and placed in an incubator, after 24 hours, the coverslip was washed with PBS, floating cells on the coverslip were removed, the washed coverslip was placed in a petri dish, a clean culture solution was added, and the petri dish with the coverslip was placed on an atomic force microscope sample stage. The method comprises the steps of imaging a cell in a contact mode, setting an indentation point on the cell in a force spectrum mode, and obtaining an indentation curve on the same indentation point by using two different acting forces. And reading indentation curve data by using data analysis software, taking indentation depth obtained by small acting force as an initial calculation point, and eliminating the influence of the substrate on the Young modulus calculation result by using a cross-correlation algorithm to obtain the elastic characteristic determined by the cytoskeleton.
Example 2:
FIG. 5 shows the indentation depth-Young's modulus curve before and after correction according to the method of the present invention, cells were inoculated onto a coverslip and placed in an incubator, after 24 hours, the coverslip was rinsed with PBS, floating cells on the coverslip were removed, the rinsed coverslip was placed in a petri dish, a clean culture solution was added, and the petri dish with the coverslip was placed on an atomic force microscope sample stage; imaging the cells in a contact mode, setting indentation points on the central position and the edge position of the cells respectively, and applying two different acting forces on the same indentation point to obtain an indentation curve; and reading indentation curve data by using data analysis software, taking indentation depth obtained by small acting force as an initial calculation point, eliminating the influence of the substrate on the Young modulus calculation result by using a cross-correlation algorithm, obtaining an indentation depth-Young modulus curve of the cell center position and the edge position, obtaining elastic characteristics determined by the cell skeleton, and obtaining the distribution condition of the cell skeleton at the center position and the edge position.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for measuring Young's modulus of single cells based on atomic force nanoindentation is characterized by comprising the following steps: in the atomic force microscope contact mode, nano indentation is carried out to measure the cell elasticity, and the method comprises the following steps: (1) Firstly, a group of indentation curves are obtained on a blank substrate, and indentation points are respectively arranged at the center position and the edge position of the cell in a 3X 3 array mode; (2) Setting different acting forces on the same indentation point to obtain different indentation depths; (3) The data segment with higher correlation coefficient between the cell force-displacement curve and the basal force-displacement curve is found by using a cross-correlation algorithm, the data segment is deleted, the data segment is compensated, and the rest data segment is prolonged to the original length, so that a corrected force-displacement curve corrected by the cross-correlation algorithm is obtained; (4) And using a two-point method of the Hertz contact model, taking the indentation depth obtained by small acting force as an initial calculation point, analyzing and calculating a cell force-displacement curve and a correction force-displacement curve, and obtaining a curve graph of Young modulus changing along with the indentation depth.
2. The method of claim 1, wherein step (1) sets the indentation points at the center and edge positions of the cell, wherein: the distribution of the intracellular cytoskeleton is determined by studying the difference of the Young's modulus of cells at different positions.
3. The method of claim 1, wherein step (2) sets forces of different magnitudes at the same indentation point, wherein: the effect of the substrate on the Young's modulus measurement of the cells was determined by studying the indentation depth of the cells by the different forces.
4. The method of claim 1, wherein step (3) uses a cross-correlation algorithm to find a data segment with a higher correlation coefficient between the cell force-displacement curve and the basal force-displacement curve, and is characterized in that: the Young's modulus of cells irrelevant to the substrate is determined by a cross-correlation algorithm, and the influence of the substrate is eliminated.
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| CN101482473A (en) * | 2009-02-13 | 2009-07-15 | 清华大学 | Method for accurately measuring Young's modulus of nano wire |
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| CN104655879A (en) * | 2015-03-05 | 2015-05-27 | 北京大学第三医院 | Method for detecting stiffness of cervical exfoliated cells by AFM (atomic force microscopy) |
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| WO2019006188A1 (en) * | 2017-06-30 | 2019-01-03 | The Regents Of The University Of California | Quantitative deformability cytometry: rapid, calibrated measurements of cell mechanical properties |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6993959B2 (en) * | 2003-10-17 | 2006-02-07 | National Institutes Of Health | System and method for the analysis of atomic force microscopy data |
| US20170299571A1 (en) * | 2016-04-14 | 2017-10-19 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Compositions and methods for determining mechanical properties of cells |
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Patent Citations (5)
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|---|---|---|---|---|
| CN101482473A (en) * | 2009-02-13 | 2009-07-15 | 清华大学 | Method for accurately measuring Young's modulus of nano wire |
| CN202057559U (en) * | 2011-04-28 | 2011-11-30 | 吉林大学 | In-situ micro-nanoscale indentation testing device based on double-displacement detection |
| CN104655879A (en) * | 2015-03-05 | 2015-05-27 | 北京大学第三医院 | Method for detecting stiffness of cervical exfoliated cells by AFM (atomic force microscopy) |
| CN107045075A (en) * | 2017-03-31 | 2017-08-15 | 国家纳米科学中心 | A kind of characterizing method of the cell physical state based on porous media model |
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