Atomic Force Microscopy Measuring Methods For Cellular/Subcellular Mechanical Properties

With the deepening research,the exploration of the pathogenesis and transfected mechanism from the cellular level has been difficult to meet the needs of current scientific research,and the exploration of the subcellular level provides a new way of thinking for the study of cell characteristics.However,there are still difficulties in the study of cellular/subcellular mechanical properties,such as poorly targeted measurement methods,single measurement parameters,and low measurement accuracy.So this work has carried out research on the Atomic force microscopy(AFM)measurement technology of cellular/subcellular structures.Take a research route from the extracellular level to the intracellular level and from quasi-static measurements to dynamic measurements.Intensive research has been carried out such as measuring static Young's modulus of cancer cells,Young's modulus of nucleus,complex Poisson's ratio,and dynamic viscoelasticity spectra of subcellular structure.The results have successfully compensated for the shortcomings of existing cell measurements.A method for measuring the static Young's modulus of cells was studied.Analyze the change law of the Young's modulus of cell death,and propose a method for mechanical description of cell death based on ultra-soft probe technology.In the developed cell viability retainer,changes in the Young's modulus of cancer cells were measured and statistically analyzed.The gelation and death process of cancer cells was quantitatively described from the perspective of mechanics,and the validity of the proposed method was verified and the AFM cell measurement platform was stability.A method for measuring the static Young's modulus of the nucleus was studied.By analyzing the contact model of polymorphic nucleus and needle tip,a method of measuring the static Young's modulus of the nucleus with needle-tip penetration into large-scale cells was proposed.Through the design and simulation of the probe structure,a fiber probe with ultra-large aspect ratio nano-tips was developed.At the same time,an optical/fluorescent microscope dual vision system is used to accurately determine the cell status and the relative position of the tip-nucleus.The measurement of the static Young's modulus of the nucleus under different cell morphologies.The effect of cell morphology on the Young's modulus of the cells was analyzed.The research on the measurement method of cell dynamic complex Poisson's ratiowas carried out.A method for measuring the dynamic complex Poisson's ratio of cells by parallel-plate tension-compression-shear simultaneous loading with magnetic drive was proposed.A dynamic Poisson's ratio measurement model based on a parallel-plate tip was established.A magnetic drive parallel plate probe was prepared to solve the problem of dynamic force coupling.The feasibility of the proposed method was verified by static Poisson's ratio test,and the effect of frequency change and indentation depth change on the value of complex Poisson's ratio was analyzed.The research of dynamic viscoelasticity spectra measurement of subcellular structures was carried out.Based on the analysis of the principle of dynamic viscoelasticity,a dynamic mechanical model of the nano-needle tip-subcellular structure was constructed,and then a method of dynamic viscoelasticity measurement of subcellular structure was proposed.A high-frequency magnetic-drive nanoneedle probe was designed and developed.The dynamic viscoelasticity spectrum was used to describe the viscoelasticity of the whole process of the nanoneedle penetrating through the subcellular structure.At the same time,analyze the characteristics of dynamic viscoelastic spectra of subcellular structures and the effect of cell status on the spectra.In addition,a multidimensional mechanical phenotypic evaluation method is proposed,and multiple mechanical phenotypes of three exemplary cells(He La,Si Ha,NIH3T3)were obtained.It makes up for the shortcomings that the static mechanical characterization method cannot distinguish homologous cancer cells,and it fully prove the feasibility,practicability and potential application value of the method.In summary,measuring the static Young's modulus of the cells,Young's modulus of the nucleus,complex Poisson's ratio,and dynamic viscoelasticity spectra of subcellular structures.The measurement of the mechanical properties of the subcellular structures provides a new method and a new system,and has important scientific research value and application prospects in the fields of disease diagnostics and biological cytology.