Development Of Broadband Nanomechanical Spectroscopy Of Live Biological Samples Via Atomic Force Microscopy

Through combining modern scientific and technological achievements such as weak signal detection,high-speed data acquisition,digital signal processing,automatic control as well as precision machinery,Atomic Force Microscopy?AFM?is becoming one of the key points of nanotechnology,which enables atomic scale imaging,surface functional studies,intermolecular force measurement,and controllable atomic&molecular operation and so on.AFM is also one of the important facilities for the study of cell biology and molecular biology.It can not only image the surface of a single living cell under physiological conditions with extremely high resolution,but also can achieve nanomechanical spectroscopy study and quantitative test on living cells.However,the existing commercial AFM systems generally have the defects of low scanning speed,narrow excitation bandwidth and prone to damage to biological samples.So it cannot meet the needs of cutting-edge research in cells and subcellular biology.This dissertation researches on broadband measurement technology of cell nanodynamic properties,and proposes a new kind of control technology to improve the measurement accuracy of AFM system.The experiment results prove the effectiveness of the study.Meanwhile,this study provides a new way for the further research on related topics of nanobiology.The main content of the thesis is divided into three parts as follows.1.A Control-based Broadband Nanoindentation?CBN?method for accurate indentation measurements of live biological samples are discussed.The method is based on the improved data-driven Modeling-free Inversion-based Iterative learning Control?MIIC?technique,which uses the hard reference sample to overcome the relative acceleration effect of the cantilever probe and minimizes the sample hydrodynamic effect on the measurement.The effect is to accurately quantify the indentation when the excitation frequency varies from 0.1Hz to 100Hz?up to four orders of scope?,while ensuring the physiological activity of the cells,significantly improving the accuracy and speed of AFM indentation measurement of live biological samples.2.A time-varying analysis method based on broadband dynamic frequency response to measure the cellular elastic and viscoelastic is proposed.Band-limited white noise excitation force was adopted to measure the nanomechanical properties of living cells at high speed?within the same frequency range,the measurement time is shorter?with wide band?1Hz¹00Hz?and deep indentation?up to several hundred nm?.By capturing reliably the dynamic response of cytoskeleton to external stimuli,real-time monitoring and quantitative analysis of the dynamic characteristics of living cells such as elastic modulus and loss modulus are realized.This provides new ideas for the study of related issues in the life sciences.3.An optimized high-speed computing framework for online real-time feedback control systems is proposed.The framework uses the Optimal Time-Distributed FFT/Time-Distributed IFFT algorithm?OTD-FFT/TD-IFFT?to improve the online computational efficiency of the classic FFT/IFFT.By means of assigning the calculation of the data sequence to different sampling periods,the computational delay and performance can be kept while the computational complexity per sampling period is effectively reduced,thereby,higher closed-loop sampling period can be achieved on the same hardware platform.The increase of sampling frequency improves significantly the speed and accuracy of online real-time feedback control system.The experimental AFM system based on the new algorithm framework can track the 800Hz high-speed triangular wave trajectory with a relative error of only 5.44%?more than 4 times lower than the classical FFT+IFFT algorithm system?.The algorithm framework can effectively improve the tracking speed and accuracy of MIIC technology,thereby improving the efficiency of cantilever deflection trajectory tracking on hard reference samples in real-time broadband monitoring experiments of nanomechanical properties of living cells.In this thesis,the methods mentioned above are applied to the nanobiology projects‘Study of Cholesterol Repletion Effect on Nanomechanical Properties of Cells',and‘Study on the dynamic characterization of broadband viscoelastic of cells'.Based on the test verification and data analysis,the following conclusions are obtained.1.Cholesterol Repletion Effect on Nanomechanical Properties of Cells.Compared with mormal human umbilical vein endothelial cells?EA.hy926?,both the Young's modulus and the complex modulus of EA.hy926 cell under the effect of cholesterol repletion were increased over 30%,respectively.Moreover,the amplitudes of both the elasticity oscillation and the viscosity oscillation at a period of around 200s were increased over 70%,respectively.The effect of cholesterol repletion on Young's modulus may be due to the fact that high cholesterol destroys the integrity of the cell membrane,and the effect of cholesterol on cell myosin activity leads to changes in vibration parameters.The results of this experiment reveal the correlation between cholesterol and myosin and subcellular cytoskeletal organization in cells,and prove the validity of the method proposed in this thesis.2.Effect of myosin on nanomechanical properties of cells.The real-time monitoring results show that the mechanical parameters of human prostate cancer cells?PC-3?are closely followed the power law.The oscillation of the dynamic viscoelastic modulus measured was periodic with a 200s period,and both the amplitude and the period of the viscoelasticity oscillation also strongly depended on the myosin activities,and closely regulated by the calcium(Ca²⁺)density of the cytoskeleton.The experiment characterizes the dynamic evolution of cell viscoelasticity,and provides a new research idea for revealing the relationship between myosin activity and cell tissue movement,and provides valuable experiments data for future research on diseases and drugs.