Method for studying whole surface mechanics of a single adherent cell under physiological conditions

Abstract

The application discloses a method for researching a whole surface mechanical atlas of a single adherent biological cell under physiological conditions, and belongs to the technical field of microscopic imaging testing. The method is based on the principle of nanoindentation technology under physiological conditions, and uses an optical microscope to perform coarse positioning on a sample to be tested, and selects the sample and sets a grid standard according to requirements. Each square of the grid is used as a basic unit, and mechanical property testing is performed on the position of each center point, and finally the testing results are output in the form of a grid pixel, so that the whole surface mechanical atlas of the adherent cell is obtained. The purpose of the application is to obtain the mechanical information of the whole cell surface under physiological conditions by using microscopic manipulation technology, and to output the analysis results in the mode of a pixel map, so that people can more intuitively understand the adhesion force difference of the whole cell surface, and a method for testing results is provided, which is more intuitive, simple and comprehensive for the in-depth research on the physical properties of cells.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting the mechanical properties of cell surface under physiological conditions, in particular to a method for researching the whole surface mechanical atlas of adherent cells under physiological conditions. BACKGROUND

[0002] The mechanical properties of cell surface are closely related to the behavior of cells. Changes in physiological activities such as cell migration, proliferation, differentiation, adhesion, and viral invasion will cause changes in the viscoelasticity of cells. Therefore, the study of the mechanical properties of cell surface helps to better understand the mechanisms of various physiological behaviors of cells [1] . As a tool for biological cell mechanics testing, the commonly used micro-manipulation methods at present include atomic force microscope technology, magnetic tweezers technology, optical tweezers technology, and micro-pipette aspiration technology. Atomic force microscope technology studies the surface structure and mechanical properties of materials through the atomic interaction force between the probe tip and the sample surface atoms when they are close enough [2] . Magnetic tweezers apply force to magnetizable beads in a gradient magnetic field to observe and analyze their movement [3] . It has the advantages of low cost and relatively simple operation, but when manipulating objects, it is necessary to add magnetic beads to the manipulated objects, which may cause damage or contamination to the sample. Optical tweezers use a strongly focused beam of light to capture objects in the micrometer range [4] . However, due to the high requirement for laser focusing, it may cause optical damage to cells, which affects the experimental results to some extent. Micro-pipette aspiration technology tests the relationship between the length of the cell elongated in the micro-pipette and the applied force by applying pressure to the micro-pipette aspiration device, thereby obtaining the mechanical properties of biological cells [5] . It can study the mechanical properties of single cells, but its precision is not high, and it is easy to damage cells during aspiration, and requires operators to have certain experience.

[0003] In biological cell manipulation, atomic force microscope technology is one of the most commonly used methods. It not only can obtain high-resolution surface topography features of biological samples under physiological conditions, but also can perform simple two-dimensional manipulation of samples, and can quantitatively design the loading process. It is widely used due to its advantages such as large force measurement range and rapid feedback to changes in mechanical information [6]Nanoindentation technology is to make the probe approach and press into the target cell under a certain load, and then unload it to separate from the target cell. In this process, the force-displacement curve between the probe and the cell can be obtained by the relationship between interatomic force and the change of the distance between the probe and the cell, and the mechanical properties of the target cell can be tested. Through this method, the mechanical information of a certain point of the cell can be tested, and for the entire cell surface, it needs to be tested point by point. After scanning the entire surface into an image, it is differentiated into a grid structure according to its topographic features, so that the probe can be pressed into the indentation point by point according to the set structure, all information is collected to extract the mechanical property parameters, and it is converted into pixel form to present, so as to obtain the complete mechanical atlas of the cell surface. The mechanical atlas can directly reflect the mechanical properties of the entire upper surface of a single cell under physiological conditions, improve the timeliness of a single experiment, avoid the randomness of the indentation technology in testing mechanical information, and its results are convenient for further mechanical performance research, and can provide a reliable theoretical basis for improving the cell adhesion force model.

[0004] [1] Scott O N, Begley M R, Komaragiri U, et al. Indentation of Freestanding Circular Elastomer Films Using Spherical Indenters. Acta Materialia, 2004, 52(16): 4877-4885.

[0005] [2] Fotiadis D, Scheuring S, Muller S A, et al. Imaging and manipulation of biological structures with the AFM. Micron, 2002, 33(4): 385-397.

[0006] [3] Vlaminck I D, Dekker C. Recent Advances in Magnetic Tweezers. Annual Review of Biophysics, 2012, 41(1): 453-458.

[0007] [4] Li Y, Lei G, Li D, et al. Progress in Optical Tweezers Technology [Invited]. Chinese Journal of Lasers, 2015, 42(1): 1-20.

[0008] [5] Trickey W R, Lee G M, Guilak F. Viscoelastic Properties of Chondrocytes from Normal and Osteoarthritic Human Cartilage. Journal of Orthopaedic Research, 2000, 18(6): 891-898.

[0009] [6] Tian Xiaojun, Wang Yuexiao, Dong Zaili, et al. Review of robotized nanomanipulation system based on AFM. Journal of Mechanical Engineering, 2009, 45(6): 14-23. SUMMARY

[0010] The present application solves the problem: in view of the deficiency of the existing mechanical property test and analysis method, a physiological condition of a single adherent cell whole surface mechanical mapping method is provided, which can characterize the whole surface mechanical properties of the cell and obtain the topographic image of the cell surface, and the method can intuitively display the whole surface mechanical properties of the adherent cell on the high-resolution space and subcellular level.

[0011] The technical solution of the present application: the technical content of the physiological condition of a single adherent cell whole surface mechanical mapping method includes: an optical microscope (1), a micro-manipulation system (2), a nanoindentation technology (3), a grid setting (4), a point-by-point mechanical property test (5), an adhesion force extraction algorithm (6), a pixel light and dark setting (7), and a grid pixel mechanical mapping (8).

[0012] The optical microscope (1) is used for rough positioning of the sample.

[0013] The micro-manipulation system (2) is used for testing the surface topography of the sample and determining the size and shape of the sample.

[0014] The nanoindentation technology (3) is used for indentation experiment on a certain point on the sample to obtain the force-displacement curve between the probe and the cell, and complete the mechanical property test of the target cell.

[0015] The grid setting (4) is used for determining the sampling grid number of the sample surface, and the result will affect the number of sampling points and the speed of the test.

[0016] The point-by-point mechanical property test (5) is used for mechanical indentation test on the center point of the selected grid.

[0017] The adhesion force extraction algorithm (6) is used for extracting the adhesion force information of the mechanical curve of all test points.

[0018] The pixel light and dark setting (7) is used for distinguishing the force value range of different area segments.

[0019] Grid pixel mechanics atlas (8), the pixel light and dark color corresponding to the force value of different regional segments is presented in the form of force spectrum.

[0020] A method for researching the whole surface mechanics atlas of a single adherent cell under physiological conditions, the implementation steps are as follows:

[0021] a. Coarse positioning of the biological sample is performed by using an optical microscope;

[0022] b. The sample is scanned by using a micro-manipulation system, the size and shape of the cell are determined, and the test range is grid set according to the parameter information, for example, 8x8;

[0023] c. The center point of the set grid is tested for mechanical properties by using the nanoindentation technology in the micro-manipulation system;

[0024] d. The mechanical curve adhesion information of all test points is extracted, and the results are classified according to different regional segment values, and the same region is a fixed light and dark color pixel;

[0025] e. The obtained different light and dark color pixels are output in the form of a grid, and finally the mechanics atlas of the surface of the sample is obtained.

[0026] Compared with the prior art, the present application has the advantages that: the present application is no longer limited to the characterization of the mechanical properties of a single point on the surface of a cell under physiological conditions, but realizes the imaging of the whole surface mechanics atlas of a single adherent cell. The results not only can obtain the mechanical information of each point, but also can intuitively reflect the mechanical properties of the whole upper surface of a single cell under physiological conditions, improve the timeliness of the experiment, avoid the randomness of the indentation technology test mechanical information, the results are convenient for further mechanical property research, and can provide reliable theoretical basis for perfecting the cell adhesion force model. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a research process schematic diagram of the method for researching the whole surface mechanics atlas of a single adherent cell under physiological conditions.

[0028] Figure 2 (a) is a principle diagram for measuring force-displacement curve by using nanoindentation technology, and (b) is a measured force-displacement curve diagram, wherein A is a needle insertion curve, and B is a needle withdrawal curve.

[0029] Figure 3 It is a schematic diagram of simulating the whole surface mechanics atlas of a single adherent cell (taking 8x8 grid as an example), wherein 1 is a frame selection range and grid setting, 2 is a probe loading route, and 3 is a measured cell.

[0030] Figure 4The images show the full-surface morphology of SMCC-7721 liver cancer cells and the corresponding mechanical atlas obtained using the method of this invention. (a) is the morphology of the target cells, and (b) is the mechanical atlas corresponding to the target cell image.

[0031] Figure 5 The images show the full-surface morphology of A549 lung cancer cells and the corresponding mechanical atlas obtained using the method of this invention. (a) is the morphology of the target cell, and (b) is the mechanical atlas corresponding to the target cell image. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings.

[0033] like Figure 1 As shown, the technical content of the method for studying the full surface mechanical map of a single adherent cell under physiological conditions according to the present invention includes: optical microscope 1, micromanipulation system 2, nanoindentation technology 3, grid setting 4, point-by-point mechanical property testing 5, adhesion force extraction algorithm 6, pixel brightness setting 7, and grid pixel mechanical map 8. The optical microscope 1 is used to coarsely locate the biological sample. After determining the sample position, the micromanipulation system 2 is used to scan the sample to obtain a morphological image of the sample. The test range is set according to the obtained cell morphology information, and the grid size of the test range is set, for example, 8×8. The nanoindentation technology in the micromanipulation system is used to test the mechanical properties of the set grid center point, such as... Figure 2 As shown in (a), the probe is subjected to a certain load to approach and press into the target cell, and then unloaded to separate it from the target cell. During this process, the force-displacement curve (hereinafter referred to as the mechanical curve) between the probe and the cell can be obtained by observing the relationship between the interatomic forces and the change in the probe-cell distance. Figure 2 (b) As shown, the mechanical properties of the target cells were tested. Adhesion force information from the mechanical curves of all test points was extracted, and the analysis results were classified according to different region segments, with each region represented by a fixed brightness and darkness pixel. The obtained pixels of different brightness and darkness were output in a grid format to finally obtain the mechanical spectrum of the sample surface.

[0034] Figure 3 This is a schematic diagram simulating the full-surface mechanical properties of a single adherent cell. After scanning the sample using a micromanipulation system, a grid is set within the scanning area according to the required data volume, determining the number and size of the grids to cover the entire cell surface. Figure 3For an 8x8 grid example, which covers the entire surface of the cell. In the test process, each cell as the basis for the work unit, and in the S-shaped order to load, in the center of each cell to complete a complete indentation experiment. According to the force-displacement curve, the mechanical information is analyzed and calculated, and the results obtained by each sampling are classified according to different regional segment values. Assign different light and dark pixels to different regional segment values, so that the image is finally presented in the form of pixels, and the mechanical atlas of the whole surface of the adherent cell is obtained.

[0035] Example 1:

[0036] As Figure 4 To realize the topographic map and the corresponding mechanical atlas of the whole surface of a single adherent cell under physiological conditions by using micro-manipulation technology. The target sample, liver cancer cells SMCC-7721, is scanned by using a micro-manipulation system, and the topographic map of the cell surface is obtained, as shown in Figure 4 (a). According to the topographic map, the grid size is set to 16x16, and a complete indentation experiment is performed at the center of each grid by using the indentation technology. The mechanical information of each point is obtained by analysis and calculation. Since the adhesion force of the cell is generally not more than 1 nN (nanonewton), and according to the data result analysis, the adhesion force value less than 400 pN (piconewton) is the darkest color, and the mechanical value increases by 100 pN to reduce one color, i.e. 400-500 pN is the second darkest color, and so on. The adhesion force value greater than 900 pN is the brightest color. According to the obtained mechanical information and the light and dark pixel assignment of different regional segment values, the mechanical atlas of the SMCC-7721 cell is obtained, as shown in Figure 4 (b). From the atlas, we can see that the adhesion force of the SMCC-7721 cell surface is basically concentrated in the 0-400 pN, 400-500 pN, and 500-600 pN intervals.

[0037] Example 2:

[0038] As Figure 5 To realize the topographic map and the corresponding mechanical atlas of the whole surface of a single adherent cell under physiological conditions by using micro-manipulation technology. The target sample, liver cancer cells SMCC-7721, is scanned by using a micro-manipulation system, and the topographic map of the cell surface is obtained, as shown in Figure 5(a) shown. According to the topography map, the grid size is set to 16x16, and the indentation technique is used to complete a complete indentation experiment for each grid center position, and the mechanical information of each point is obtained according to the analysis and calculation. Since the adhesion force of the cell is generally not more than 1nN (nanonewton), and according to the data result analysis, the adhesion force value less than 200pN (piconewton) is set to the darkest color, and then the mechanical value is reduced by one color density every 100pN, that is, 200-300pN is the second dark color, and so on. The adhesion force value greater than 1nN is the brightest color. According to the obtained mechanical information and the light and dark pixel assignment of different regional segment values, the mechanical map of the A549 cell as shown in Figure 5 (b) shown. From the map, we can see that the adhesion force of the A549 cell surface is basically concentrated in the 0-200pN, 200-300pN, and 300-400pN intervals.

[0039] From the above experimental results using the method of the present application, it can be seen that the method not only can obtain the mechanical information of each point, but also can intuitively reflect the mechanical characteristics of the entire upper surface of a single cell under physiological conditions, which can provide a theoretical research basis for the future research of cell surface protein mechanics, drug efficacy evaluation, and pathological analysis. The method improves the timeliness of the existing experiment, avoids the randomness of the indentation technique test mechanical information, and its results are convenient for further mechanical property research, and can provide a reliable theoretical basis for perfecting the cell adhesion force model.

[0040] The part of the present application not described in detail belongs to the known technology in the art.

[0041] The above is only part of the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for studying the full surface mechanical map of a single adherent cell under physiological conditions, characterized in that : Comprising the steps of: a. Coarsely positioning the biological sample using an optical microscope; b. Scanning the sample using a micromanipulation system, determining the size and shape of the cells, and setting a grid for the test range according to the parameter information; c. Point-by-point mechanical property testing of the set grid center point using nanoindentation technology in the micromanipulation system; d. Extracting the mechanical curve adhesion information of all test points, and classifying the results according to different regional segment values, with the same region being a fixed light and dark pixel, and using the light and dark of the pixel to present the molecular point mechanics size distribution of the whole surface of the adherent cells; e. Outputting the obtained different light and dark pixels in a grid form, and finally obtaining the grid pixel mechanics atlas of the sample surface; the grid pixel mechanics atlas of the whole surface of the adherent cells can be obtained in a single test, and rapid analysis of the cell surface mechanics is realized.

2. The method for mapping the full-surface mechanical properties of a single adherent cell under physiological conditions according to claim 1, characterized in that: According to the size and shape of different biological samples, the grid setting for the mechanical test range is more targeted.

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