Study On The Optimization Of 3D Super-resolution Fluorescence Microscopy And Its Biological Application

Optical microscopy especially fluorescence microscopy is the most commonly used tools in life science, which has some outstanding advantages, such as nondestructive, contactless,high specificity, high sensitivity, high friendly with living cells and able to provide function information. However, the spatial resolution of conventional optical microscope is classically limited by the diffraction of light to ~200 nm and ~500 nm in the lateral and axial directions,respectively. Over the past several years, this diffraction limit has been overcome with the development of the far field super resolution fluorescence microscopy approaches that can achieve resolutions of about 10-20 nm in all three dimensions. So we can observe the intracellular hyperfine structure, dynamic process and function in living cells at a truly molecular-scale resolution level. This technology will lead to unprecedented visualization and deeper understanding of various life process, thereby greatly promote the development of life sciences and other fields. The work presented here concentrates on single molecule localization based super-resolution fluorescence microscopy and its application to study of lipid rafts.Although single molecule localization based super-resolution fluorescence microscopy has made great progress, there are still challenges to be faced for live-cell imaging. For instance, how to further improve the spatial and temporal resolution of the system to achieve rapid nanometer resolution imaging of living cells. The photoswitching properties of fluorescent molecule and the drift of imaging system are the key technical factors that can affect the spatial and temporal resolution of the system to be improved furtherly. In order to solve these problems, this thesis is focused on the buffer preparation procedure and drift correction methods for 3D-STORM. The main work of this paper are listed as follows:1. Designed and finished a contract experiment with buffers. Through experiment, we find a suitable buffer that can be used for the 3D-STORM system commendably. The results show that the buffer can control the sparse extent of excited fluorescence molecules better than ever, and also improve the stability of blinking of the fluorescent molecules.2. Researched two methods of drift correction: using fiduciary markers to correct driftsand using bright field images to correct drifts. A modified redundant cross-correlation method of drift correction is rededuced and designed. The experiment results show that the transverse correction accuracy of the drift correction algorithm that based on coordinate-correction reached 10 nm, which can simplify the experiment process without adding any optical structure to the original system. The drift correction algorithm also can improve the efficiency of data processing.3. Designed a specific marker method of lipid rafts in the cell membrane, and then used3D-STORM system to record and reconstruct a nanometer image of cell lipid raft samples.The experiment results show that the lipid rafts’ GM1 in the Alzheimer’s disease cell membrane are more sequential and compact than the normal cell, and the lipid rafts’ quantity are more than normal cell. This experiment also provided more intuitive information about the lipid rafts in the cell membrane.