Scanning Probe Acoustic Microscopy Imaging And Characterization Of Cells

With the rapid development of nanotechnology in biomedicine,it is important to image and characterize cells and physiological phenomena in the cellular microenvironment at the nanoscale by nondestructive testing.The traditional ultrasonic imaging and optical imaging technologies suffer low resolution due to the limitation of diffraction.SPAM(Scanning Probe Acoustic Microscope)is a technique that combines AFM(Atomic Force Microscope)with ultrasonic nondestructive technology.This technique can not only achieve ultra-high resolution imaging of the sample surface and substructure,but also analyze and characterize the nano-mechanical properties.It has a wide range of applications.This thesis focuses on the theoretical analysis,system construction,and its applications in imaging and characterization of cells and cell-substrate interactions.The main work and results are as follows:1.Based on the working principle of SPAM and combining with the dynamic properties of cantilever,the system characteristics,signal generation and imaging mechanism of SPAM were analyzed.According to the dynamic analysis of the tip-sample interactions,the dynamic stress process of the tip on elastic and viscoelastic surfaces was simulated,and the characteristic equation of the cantilever system under contact state was solved.The relationship between the contrast of acoustic image and the stiffness of material was analyzed,the relationship between the mechanical property changes caused by embedding other materials into internal materials and the cantilever vibration signal was established,and the amplitude and phase variations were analyzed.2.An SPAM system for the study of biological cells was designed and constructed.The imaging characteristics of SPAM in air and liquid were analyzied,and a special air/liquid scanning probe and probe holder were developed.A new optical tracking method of SPAM is designed,which can detect the probe movement in whole liquid mode and in air.It was designed a focusing lens group and a glass window on the probe frame to eliminate the influence of surface wave and refraction of liquid.Considering the characteristics of biological cells,the system structure design,hardware construction and software control were carried out,and the system was used for imaging live cells in liquid.Furthermore,SPAM was used to study the defect detection and surface structure of liver cancer cells in air.In addition,the SPAM imaging affected factors(including tip wear,phase and sensitivity)were analyzed and discussed.3.The effects of patterned stiffness and topographic substrates on cell behaviors were studied based on SPAM.The surface of SU-8 thin film was modified by electron beam exposure technology to produce nano patterns with designed stiffness and morphology.SPAM was used to image and characterize the surface morphology and stiffness distribution of the modified SU-8 substrates.The acoustic images showed structures notshown in the morphology images.Fluorescence microscope and SPAM were used to study the responses of cell behaviors on the different substrates.Compared with fluorescence image and topography image,the relationship between mouse fibroblasts(L929)and the substrate could be easily observed by acoustic image.The results showed that topography has a more significant effect on cell behaviors than stiffness on patterned substrates on nanoscale.4.The subsurface structure of mouse fibroblasts(L929)on bionic nanoarrays was studied based on SPAM.Hybrid silicon nanowire and silicon nanopore arrays were developed on the silicon substrate as a biomimetic surface using laser interference lithography and metal-assisted chemical etching.SPAM and scanning electron microscope(SEM)were used to observe and analysis the L929 cells responses after cultured on the nanoarrays,and the results of the two imaging technologies were compared to evaluate the imaging capability of SPAM on the cell-substrate surface and subsurface.The results showed that SPAM could not only obtain the structural image,but also judge the structural changes of the object by the signal intensity.Furthermore,the morphology of L929 cells on the surface of the hybrid nanoarrays was analyzed,and it was found that L929 cells could perceive the morphology of the hybrid nanoarrays.This work can guide the design of biological interfaces and provide a useful tool for the study of cell subsurfaces in different biological domains.