CN-121995083-A - Method for measuring nuclear modulus based on AFM

CN121995083ACN 121995083 ACN121995083 ACN 121995083ACN-121995083-A

Abstract

The invention discloses a method for measuring nuclear modulus based on AFM, which comprises the steps of extracting a nuclear sample, combining Sneddon contact model and Maxwell viscoelastic model to obtain a needle insertion curve formula, measuring separated cell nuclei by using an atomic force microscope, and processing experimental data by using Matlab. The method has the advantages that a force-distance curve is obtained by measuring the separated cell nucleuses by using a probe, then, a Sneddon contact model which accords with the indentation depth and is far larger than the radius of the needle point and a Maxwell viscoelastic model are combined, the relation between the cell nucleuses viscoelasticity and the influence of cell skeletons on the cell nucleuses modulus and the relation between the needle point radius and the indentation depth are considered, a formula suitable for measuring the cell nucleuses modulus is deduced, the Matlab fitting experimental data is used for obtaining the mechanical parameters of the cell nucleuses, the influence of the cell skeletons and other organelles on the cell nucleuses viscoelasticity parameters can be avoided in the measuring process, and the measuring result is closer to the real parameters of the cell nucleuses.

Inventors

ZHAO PENGXIANG
CHEN LONGQUAN
YU YUE

Assignees

电子科技大学

Dates

Publication Date: 20260508
Application Date: 20260127

Claims (7)

1. A method for measuring nuclear modulus based on AFM, comprising the steps of: s1, extracting a cell nucleus sample; S2, combining Sneddon with the contact model and the Maxwell viscoelastic model to obtain a needle insertion curve formula; s3, measuring the separated cell nuclei by using an atomic force microscope; S4, processing experimental data by using Matlab.
2. A method of measuring nuclear modulus based on AFM according to claim 1, the method is characterized in that the step S1 specifically comprises the following steps: Adherent cells cultured in a petri dish were subjected to a membrane melting treatment with Triton X-100 at a concentration of 1%, and the cell sap was taken away and added to Triton X-100 and left to stand at room temperature for 20 minutes, followed by taking off Triton X-100 and washing with PBS twice to ensure complete removal of Triton X-100 and storage with PBS.
3. A method of measuring nuclear modulus based on AFM according to claim 1, the method is characterized in that the step S2 specifically comprises the following steps: obtaining Senddon a model formula and a 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, v is the poisson ratio; the Maxwell viscoelastic model formula is, Η is the viscosity parameter, E1 and E2 are the elasticity parameters, τ=η/E2 is the relaxation time; superposing the Senddon model formula and the Maxwell viscoelastic model formula to obtain a final needle insertion curve formula, T is the time at which the indentation is the deepest.
4. A method of measuring nuclear modulus based on AFM according to claim 1, the method is characterized in that the step S3 specifically comprises the following steps: S31, setting an atomic force microscope and calibrating, namely preparing a probe with the radius of the probe tip of 63nm, adjusting the position of the probe after the probe is loaded by the atomic force microscope to enable laser to strike the back surface of the probe tip, and calibrating the modulus of the probe through high-frequency vibration under a liquid phase; s32, setting a contact threshold; S33, scanning the surface of the sample by using the scanning function of the AFM, and determining the position of force measurement through morphology; s34, setting the needle inserting force of the AFM, and carrying out multipoint mechanical measurement on the area above the cell nucleus to obtain a force distance curve.
5. A method of measuring nuclear modulus based on AFM according to claim 1, the method is characterized in that the step S4 specifically comprises the following steps: Exporting data after the experiment is completed, and performing batch fitting on the data by using a program written by Matlab to obtain a result; and setting a parameter range to screen the data.
6. The method of claim 4, wherein the contact threshold is 30pN, the needle insertion force is 500pN-1000pN, and the upper region is defined as a region of 5um x 5 um.
7. The method of claim 5, wherein the derived data comprises needle penetration distance, needle penetration time, and needle penetration feedback force data.

Description

Method for measuring nuclear modulus based on AFM Technical Field The invention relates to the field of cell mechanics, in particular to a method for measuring nuclear modulus based on AFM. Background The cell nucleus is taken as the largest organelle in the cell, DNA replication and mRNA transcription carried out in the cell nucleus control the growth of the cell and the synthesis of proteins, abnormal cell nucleus and chromatin structure are important markers of various diseases, mutation of structural proteins Lamins A of a cell nuclear membrane can lead to premature senility, the rigidity of the cell nucleus is obviously increased, the developing nerve tissue lacks the expression of a layer Lamins A/C, the rigidity of the cell nucleus is reduced, the nuclear membrane can be broken when the cell is in an extrusion environment, substances in the cell cytoplasm can enter the cell nucleus and cause DNA damage, and thus, the cell canceration is caused, and therefore, the research on the mechanical properties of the cell and the cell nucleus is of great importance. Most of the current methods for measuring the nuclear modulus measure the nuclear modulus in the whole cell by magnetic forceps, optical forceps or microfluidic methods, and the cell nucleus is stretched and extruded by the cell skeleton, so that the deviation of the modulus measured in the whole cell can occur, and even the highest place of the whole cell directly measured by using AFM is studied as the nuclear modulus, and the measured data cannot represent the modulus of the cell nucleus because part of the cell skeleton may exist above the cell nucleus. In addition, since the cell nucleus is a viscoelastic substance, many documents currently measuring the cell nucleus only treat the cell nucleus as a pure elastic object, so that the measurement result is greatly different from the real parameter. Disclosure of Invention The invention aims to provide a method for measuring the nuclear modulus based on AFM. The invention realizes the above purpose through the following technical scheme: a method for measuring nuclear modulus based on AFM, comprising the steps of: s1, extracting a cell nucleus sample; S2, combining Sneddon with the contact model and the Maxwell viscoelastic model to obtain a needle insertion curve formula; s3, measuring the separated cell nuclei by using an atomic force microscope; S4, processing experimental data by using Matlab. Further, the step S1 specifically includes: Adherent cells cultured in a petri dish were subjected to a membrane melting treatment with Triton X-100 at a concentration of 1%, and the cell sap was taken away and added to Triton X-100 and left to stand at room temperature for 20 minutes, followed by taking off Triton X-100 and washing with PBS twice to ensure complete removal of Triton X-100 and storage with PBS. Further, the step S2 specifically includes: obtaining Senddon a model formula and a 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, v is the poisson ratio; the Maxwell viscoelastic model formula is, Η is the viscosity parameter, E1 and E2 are the elasticity parameters, τ=η/E2 is the relaxation time; superposing the Senddon model formula and the Maxwell viscoelastic model formula to obtain a final needle insertion curve formula, T is the time at which the indentation is the deepest. Further, the step S3 specifically includes: S31, setting an atomic force microscope and calibrating, namely preparing a probe with the radius of the probe tip of 63nm, adjusting the position of the probe after the probe is loaded by the atomic force microscope to enable laser to strike the back surface of the probe tip, and calibrating the modulus of the probe through high-frequency vibration under a liquid phase; s32, setting a contact threshold; S33, scanning the surface of the sample by using the scanning function of the AFM, and determining the position of force measurement through morphology; s34, setting the needle inserting force of the AFM, and carrying out multipoint mechanical measurement on the area above the cell nucleus to obtain a force distance curve. Further, the step S4 specifically includes: Exporting data after the experiment is completed, and performing batch fitting on the data by using a program written by Matlab to obtain a result; and setting a parameter range to screen the data. Further, the contact threshold is 30pN, the needle insertion force is 500pN to 1000pN, and the upper region is defined as a region of 5um x 5 um. Further, the derived data after the experiment is completed comprises needle insertion distance, needle insertion time and needle insertion feedback force data. The method for measuring the nuclear modulus based on the AFM has the advantages that a force-distance curve is obtained by measuring the separated cell nucleus by using a probe, then a Sneddon contact model whi