STORM Super-resolution Imaging Studies Of Cytoskeleton

Cells are the basic unit of life activities.Observing the distribution and interaction of different material and element inside the cells by microscopic imaging is an important mean for biological study in nowadays.There are many characteristics such as non-destructive,contactless,high specificity and sensitivity.But limited by the optical diffraction,the resolution of traditional microscopy only reaches to about 200 nm.To a large extent,it limits us to observe and study the component and function of nanoscale biological molecules.For the past few years,with the development of science and technology scientists excogitated the super-resolution microscopy that breaks through the optical diffraction limitation,and its resolution can reach to 10-50nm(get close to the resolution of electron microscope).Super-resolution microscopy which base on the far-field optical microscope mainly divided into two categories: super-resolution which uses the way of nonlinear optics include Stimulated Emission Depletion(STED)microscopy and Saturation Structure Illumination Microscopy(SSIM);super-resolution which uses light reversible molecular to gain the location of single molecules include Photoactivation Localization Microscopy(PALM)and Stochastic Optical Reconstruction Microscopy(STORM).They have developed rapidly and applied to every fields of biology such as observing membrane protein's distribution,cytoskeleton,mitochondria of cells.The study of cytoskeleton in biology is of great significance,but the diameter of the cytoskeleton is about dozens of nanometer size which is difficult to observe by conventional confocal microscope.So this paper proposes to study the skeletal structure of cells by super-resolution imaging microscopy.We take the experiments for optimizing sample's antibody concentration and incubation condition--the incubation condition of first antibody and different antibody concentration experiments;for optimizing excitation light power.Experimental results show that first antibody incubated overnight(about 12 hours)make the sufficient labeling to microtubule,and obtain more continuous structure.It has little effect on different antibody concentration,and illustrates that the label concentration has been oversaturation.Using 20 m W excitation light power obtains better reconstruction result.This illustrates that when excitation light power gets to around 20.0m W,it obtains more photon for single blinking and more continuous structure.Oxygen takes part in the cell inside cycle to produces energy for cell growth,proliferation and synthesis.Therefore oxygen is one of the necessary gas for cell's normal growing.Hypoxia leads to anomalous change in tissue metabolism,function and morphological structure.In the hypoxia environment,it mainly damage cytomembrane,mitochondria and lysosome.Many microenviroment of tumor tissue is in hypoxia status,and hypoxia is closely related to the physiological function of tumor cells.Then labelling the hypoxia cytoskeleton and taking the STORM imaging by theexperimental system which has been optimized,compare the super-resolution imaging figures that normal cultured cells and anoxic cells,then analyze and discuss the imaging results.It shows that the microtubule structure of hypoxia He La cells become more staggered clutter and very dense.Such situation will also appear in the cell division period stage.This result is consistent with the report of 2017 Ji?í ?ehulka group study of hypoxia tumor cells[47]: by some unknown way,cells enter the stage of cell division which causes or results the widespread diffusion of tumor cells,and enhance its resistance of medicine.