Acquisition Of Breast Cancer Characteristics At Single Cell Level By AFM Combined With Optical Microscopy

The micro/nano structure of cells changed during the procession of the tumor growth.With the rapid development of imaging techniques,the acquisition,diagnosis and treatment based on the ultra-nano characteristics of cancer cells at single cell level can become a new trend for oncology medicine.In the thesis,three breast cell lines MCF-7,MDA-MB-231 and MCF-10A were used in the experiment.Atomic force microscopy and optical microscopy including structure illustration microscopy,Raman spectroscopy and two-photon microscopy were applied to characterize the biophysical properties of the cells and investigate the features of breast cancer cells as well as the related molecular mechanism.First,Atomic force microscopy(AFM)was performed on different subtypes of the breast tumor cells,and the topographic features and nanostructure of the cell surface were obtained.The combination of AFM and SIM(strucuture illustration microscopy)was successfully constructed,and can be used to image the actin cytoskeleton of the cells.The correlative system not only can offer the information of morphology,but also can obtain the elasticity properties of breast tumor cells with different metastatis,which can offer a new imaging approach and technique for investigation of cell structure and function.Second,the biophysical properties of living cells were acquired by AFM.The influence factors on the measurement were analyzed and the optimal parameters used in the experiment were obtained.Under the optimal condition,the force curves were perfomed and then analysed with appropriate model to calculate the elasticity modulus,viscosity and adhesion force.The dependence of the biophysical properties on the actin cytoskeleton was also discussed.In addition,Raman spectroscopy was used to measure the molecular vibration spectra of three breast cell lines and LDA-PCA algorithm was applied to discriminate the characteristic spectra of the cells.The results indicate that different subtypes of breast cell lines could be obviously featured and characterized by the combination of AFM and Raman spectroscopy techniques,which is a promising approach as a clinical tool and could shed new light for early cancer discrimination at single cell level.Finally,rapid action of estrogen on biophysical properties and membrane lipid order of MCF-7 cells were investigated by AFM and two photon microscopy.The results show that the biophycial properties including cell force,cell-surface roughness,elasticity and viscosity decrease rapidly after the addition of estrogen at the appropriate concentration.The alteration of biophysical properties may attribute to actin reorganization of the cells.This rapid signaling may be mediated by estrogen receptors localized to plasma membrane and can induce the alteration of membrane lipid order which can represent the lipid raft characterization.The results suggest that lipid raft may be involved in estrogen-trigerred non-genomic effects.It is hoped that further investigation of such phenomena will help to develop novel strategies and new drugs for antitumor therapy.In conclusion,the abilitiy of AFM to investigate micro-nano characteristics of living cells can be improved greatly in combination with optical microscopy.AFM aided with optical microscopy can offer multi-information of living cells,which indicates that it could be a promising technique for acquisition of breast tumor features at single cell level and could help to gain insight into biological processes of tumor metastasis.