Method for measuring cell nuclear modulus based on AFM

Abstract

The invention discloses a method for measuring a cell nucleus modulus based on an AFM (atomic force microscope). The method comprises the following steps: extracting a cell nucleus sample, obtaining a needle insertion curve formula by combining a Sneddon contact model and a Maxwell viscoelastic model, measuring separated cell nucleuses by using an atomic force microscope, and processing experimental data by using Matlab. The method has the beneficial effects that a force-distance curve is obtained by measuring separated cell nucleuses by using a probe, and then in combination with a Sneddon contact model and a Maxwell viscoelasticity model, the influence of cell nucleus viscoelasticity and cytoskeleton on cell nucleus modulus and the relationship between the radius of the needle tip and the indentation depth are considered, so that the accuracy of the cell nucleus modulus is improved. According to the method, a formula suitable for measuring the cell nucleus modulus is deduced, the mechanical parameters of the cell nucleus are obtained by using Matlab fitting experimental data, the influence of a cytoskeleton and other organelles on the viscoelastic parameters of the cell nucleus can be avoided in the measurement process by using the measured and separated cell nucleus, and the measurement result is closer to the real parameters of the cell nucleus.

Description

Technical Field

[0001] This invention relates to the field of cell mechanics, and in particular to a method for measuring cell nuclear modulus based on AFM. Background Technology

[0002] As the largest organelle in the cell, the nucleus is where DNA replication and mRNA transcription take place, controlling cell growth and protein synthesis. Abnormalities in the structure of the nucleus and chromatin are important markers of various diseases. Mutations in the structural protein Lamins A of the nuclear membrane can lead to premature aging and a significant increase in nuclear rigidity. However, developing neural tissue lacks expression of the Lamins A / C layer, which reduces the rigidity of the nucleus. When the cell is under pressure, the nuclear membrane may rupture, allowing substances from the cytoplasm to enter the nucleus and cause DNA damage, leading to cell carcinogenesis. Therefore, studying the mechanical properties of cells and the nucleus is of great significance.

[0003] Most current methods for measuring nuclear modulus involve using magnetic tweezers, optical tweezers, or microfluidics to measure nuclear modulus within intact cells. However, the nucleus is stretched and compressed by the cytoskeleton within the cell, leading to deviations in the measurement of nuclear modulus within intact cells. Some studies have even used AFM to directly measure the highest point of an intact cell as the nuclear modulus. Since there may be some cytoskeleton above the nucleus, the measured data cannot represent the nuclear modulus.

[0004] In addition, since the cell nucleus is a viscoelastic material, and many current studies on cell nucleus measurement simply treat it as a purely elastic object, the measurement results differ significantly from the actual parameters. Summary of the Invention

[0005] The purpose of this invention is to provide a method for measuring cell nuclear modulus based on AFM.

[0006] The present invention achieves the above objectives through the following technical solutions: A method for measuring nuclear modulus based on AFM includes the following steps: S1. Extract cell nucleus samples; S2. The needle insertion curve formula is obtained by combining the Sneddon contact model and the Maxwell viscoelastic model. S3. Use an atomic force microscope to measure the isolated cell nuclei; S4. Use Matlab to process the experimental data.

[0007] Further, step S1 specifically includes: The adherent cells cultured in the culture dish were thawed using a 1% Triton X-100 solution. The cell suspension was aspirated, Triton X-100 was added, and the mixture was placed at room temperature for 20 minutes. Then, the Triton X-100 was aspirated, and the cells were rinsed twice with PBS to ensure complete removal of Triton X-100. The cells were then stored in PBS.

[0008] Further, step S2 specifically includes: Obtain the Senddon model formula and the Maxwell viscoelastic model formula, wherein the Senddon model formula is: θ is the needle tip half angle, δ is the indentation depth, E is the elastic model, and v is Poisson's ratio; The Maxwell viscoelastic model formula is as follows: η is the viscosity parameter, E1 and E2 are the elastic parameters, and τ=η / E2 is the relaxation time; By performing a Boltzmann superposition of the Senddon model formula and the Maxwell viscoelastic model formula, the final needle insertion curve formula is obtained. t represents the moment when the indentation is deepest.

[0009] Furthermore, step S3 specifically includes: S31. Set up and calibrate the atomic force microscope. Specifically, prepare a probe with a tip radius of 63 nm. After loading the probe onto the atomic force microscope, adjust the probe position so that the laser hits the back of the tip. Then, calibrate the modulus of the probe by high-frequency vibration in the liquid phase. S32. Set the contact threshold; S33. Use the scanning function of AFM to scan the sample surface and determine the location of force measurement by morphology; S34. Set the insertion force of AFM and perform mechanical measurements at multiple points in the region above the cell nucleus to obtain the force-distance curve.

[0010] Furthermore, step S4 specifically includes: After the experiment is completed, the data is exported and a program written in Matlab is used to perform batch fitting to obtain the results; Set parameter ranges to filter the data.

[0011] Furthermore, the contact threshold is 30pN, the needle insertion force is 500pN-1000pN, and the upper region is defined as a 5um*5um area.

[0012] Furthermore, the data exported after the experiment includes needle insertion distance, needle insertion time, and needle insertion feedback force data.

[0013] The beneficial effects of the method for measuring nuclear modulus based on AFM described in this invention are as follows: Force-distance curves are obtained by measuring isolated cell nuclei using a probe. Then, by combining the Sneddon contact model (where the indentation depth is much greater than the needle tip radius) and the Maxwell viscoelastic model, the influence of nuclear viscoelasticity and the cytoskeleton on nuclear modulus is considered. Furthermore, the relationship between the needle tip radius and the indentation depth is taken into account, deriving a formula suitable for measuring nuclear modulus. The mechanical parameters of the cell nucleus are obtained by fitting experimental data using Matlab. By measuring the isolated cell nuclei, the influence of the cytoskeleton and other organelles on the viscoelastic parameters of the cell nucleus can be avoided during the measurement process, resulting in measurement results that are closer to the true parameters of the cell nucleus. Attached Figure Description

[0014] Figure 1 A flowchart illustrating a method for measuring nuclear modulus based on AFM according to an embodiment of the present invention; Figure 2 Electron micrographs of GES-1 cells treated with Triton X-100 for different durations using atomic force microscopy to measure cell nuclear modulus in an embodiment of the present invention. Figure 3 Fluorescence images of GES-1 cells treated with Triton X-100 for different durations using atomic force microscopy to measure cell nuclear modulus in an embodiment of the present invention. Figure 4 A model diagram illustrating the method for measuring cell nuclear modulus based on AFM, as provided in an embodiment of the present invention; Figure 5 Force-distance curves and their fitting effect diagrams for the method of measuring cell nuclear modulus based on AFM provided in the embodiments of the present invention; Figure 6 This invention provides a comparison of various parameters between live cells and isolated cell nuclei at different needle insertion speeds in the measurement of cell nuclear modulus using atomic force microscopy, as provided in an embodiment of the invention. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] like Figure 1-6As shown, this invention provides a method for measuring cell nuclear modulus based on AFM, comprising the following steps: S1. Extract cell nucleus samples; Adherent cells cultured in culture dishes were thawed using a 1% Triton X-100 solution. The cell culture medium was aspirated, Triton X-100 was added, and the mixture was incubated at room temperature for 20 minutes. The Triton X-100 was then aspirated, and the cells were washed twice with PBS to ensure complete removal of the Triton X-100. The cells were then stored in PBS. Figure 2-3 In the study, the states of GES-1 cells varied after treatment for different durations. Figure 3 In the diagram, Dapi stains the chromatin in the cell nucleus, Cytoskeleton stains the cytoskeleton, and Lamin A / C stains the nuclear membrane. In this embodiment, different samples were obtained by treating GES-1 cells and Nih3t3 cells. When GES-1 cells were thawed using 1% Triton X-100, the cells were treated for different durations, and the thaw results were verified by fluorescence microscopy and electron microscopy.

[0017] S2. The needle insertion curve formula is obtained by combining the Sneddon contact model and the Maxwell viscoelastic model. The Senddon model is applicable when the contact radius is much smaller than the indentation depth, and it matches the actual measurement data. The Senddon model formula is as follows: Where θ is the needle tip half-angle, which is 15 degrees in this embodiment; δ is the indentation depth; E is the elastic model; and v is Poisson's ratio, which is set to 0.5 in this embodiment. However, since the cell nucleus is a viscoelastic material, the above formula needs to be modified for E, referring to the Maxwell viscoelastic model (see...). Figure 4 ), The cell nucleus was processed using the Maxwell viscoelastic model, a type of SLS model. Its intrinsic structure consists of a spring connected in series with a damper, which is then connected in parallel with another spring. Here, η is the viscous parameter, E1 and E2 are the elastic parameters, and τ = η / E2 is the relaxation time. The Senddon model formula and the Maxwell viscoelastic model formula were then superimposed using Boltzmann superposition to obtain the final needle insertion curve formula. Where t represents the moment when the indentation is deepest.

[0018] S3. Use an atomic force microscope to measure the isolated cell nuclei; S31. Set up and calibrate the atomic force microscope. Specifically, prepare a probe with a tip radius of 63 nm. After loading the probe onto the atomic force microscope, adjust the probe position so that the laser hits the back of the tip. Then, calibrate the modulus of the probe by high-frequency vibration in the liquid phase. S32. Set the contact threshold. Set the needle contact threshold to 30pN before measurement (this standard can protect the needle tip from damaging the sample). Then, use an optical microscope to lower the probe to quickly approach the sample surface. When it is close to the sample, use the automatic approach function to ensure that the probe does not directly penetrate the sample. S33. Use the scanning function of AFM to scan the sample surface and determine the location of force measurement by morphology; S34. Set the insertion force of the AFM needle and perform multiple mechanical measurements in the region above the cell nucleus to obtain the force-distance curve (refer to...). Figure 5 The needle insertion force is 500pN-1000pN, and the upper region is defined as a 5um × 5um area; In this embodiment, a loading force of 500pN and an indentation depth of 2µm were set, and force-distance curves were obtained by performing multi-point mechanical measurements on the cell nucleus surface (this standard can minimize the need for the needle tip to stick to the sample surface and become unable to detach from the sample). Reference Figure 6 In this step, the isolated cell nuclei were measured by setting four different needle insertion rates (1Hz: 5.94μm / s, 1.5Hz: 9.02μm / s, 2Hz: 12.05μm / s, 2.5Hz: 15.03μm / s). The measurement data are shown in the following four tables: Table 1 Table 2 Table 3 Table 4 Tables 1 and 2 show the values ​​of E1 and E2 at different speeds, in kPa; Table 3 shows the values ​​of τ, in s; and Table 4 shows the values ​​of η, in Pa·s.

[0019] S4. Use Matlab to process the experimental data; After the experiment was completed, the data was exported, including needle insertion distance, needle insertion time, and needle insertion feedback force. A program written in Matlab was used to perform batch fitting on the data to obtain the results. Set parameter ranges to filter the data.

[0020] In the experiment of this embodiment, due to the influence of liquid flow, the experimental data needs to be preprocessed before curve fitting to obtain viscoelastic parameters. The needle insertion curve is calibrated according to the stage before contact with the sample (baseline). After calibration, the baseline is truncated, leaving only the needle insertion process. Then, Matlab is used to write the fitting program. After curve fitting, the viscoelastic parameters are obtained. At the same time, the data is filtered according to the obtained elastic parameters, viscoelastic parameters and the sum of squared errors of the fitting, and finally the valid data is retained.

[0021] In this embodiment, force-distance curves are obtained by measuring the isolated cell nuclei using a probe. Then, combining the Sneddon contact model (where the indentation depth is much larger than the needle tip radius) and the Maxwell viscoelastic model, the influence of nuclear viscoelasticity and the cytoskeleton on nuclear modulus is considered. Furthermore, the relationship between the needle tip radius and the indentation depth is taken into account, and a formula suitable for measuring nuclear modulus is derived. The mechanical parameters of the cell nucleus are obtained by fitting experimental data using Matlab. By measuring the isolated cell nuclei, the influence of the cytoskeleton and other organelles on the viscoelastic parameters of the cell nucleus can be avoided during the measurement process, and the measurement results are closer to the true parameters of the cell nucleus.

[0022] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for measuring nuclear modulus based on AFM, characterized in that, Includes the following steps: S1. Extract cell nucleus samples; S2. The needle insertion curve formula is obtained by combining the Sneddon contact model and the Maxwell viscoelastic model. S3. Use an atomic force microscope to measure the isolated cell nuclei; S4. Use Matlab to process the experimental data.

2. The method for measuring nuclear modulus based on AFM according to claim 1, characterized in that, Step S1 specifically includes: The adherent cells cultured in the culture dish were thawed using a 1% Triton X-100 solution. The cell suspension was aspirated, Triton X-100 was added, and the mixture was placed at room temperature for 20 minutes. Then, the Triton X-100 was aspirated, and the cells were rinsed twice with PBS to ensure complete removal of Triton X-100. The cells were then stored in PBS.

3. The method for measuring nuclear modulus based on AFM according to claim 1, characterized in that, Step S2 specifically includes: Obtain the Senddon model formula and the Maxwell viscoelastic model formula, wherein the Senddon model formula is: θ is the needle tip half angle, δ is the indentation depth, E is the elastic model, and v is Poisson's ratio; The Maxwell viscoelastic model formula is as follows: η is the viscosity parameter, E1 and E2 are the elastic parameters, and τ=η / E2 is the relaxation time; By performing a Boltzmann superposition of the Senddon model formula and the Maxwell viscoelastic model formula, the final needle insertion curve formula is obtained. t represents the moment when the indentation is deepest.

4. The method for measuring nuclear modulus based on AFM according to claim 1, characterized in that, Step S3 specifically includes: S31. Set up and calibrate the atomic force microscope. Specifically, prepare a probe with a tip radius of 63 nm. After loading the probe onto the atomic force microscope, adjust the probe position so that the laser hits the back of the tip. Then, calibrate the modulus of the probe by high-frequency vibration in the liquid phase. S32. Set the contact threshold; S33. Use the scanning function of AFM to scan the sample surface and determine the location of force measurement by morphology; S34. Set the insertion force of AFM and perform mechanical measurements at multiple points in the region above the cell nucleus to obtain the force-distance curve.

5. The method for measuring nuclear modulus based on AFM according to claim 1, characterized in that, Step S4 specifically includes: After the experiment is completed, the data is exported and a program written in Matlab is used to perform batch fitting to obtain the results; Set parameter ranges to filter the data.

6. The method for measuring nuclear modulus based on AFM according to claim 4, characterized in that: The contact threshold is 30pN, the needle insertion force is 500pN-1000pN, and the upper region is defined as a 5um*5um area.

7. The method for measuring nuclear modulus based on AFM according to claim 5, characterized in that: The data exported after the experiment includes needle insertion distance, needle insertion time, and needle insertion feedback force data.

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