The Research And Application Of Stimulated Emission Depletion Microscopy

Fluorescence microscopy is an essential equipment in Biology and Chemistry lab due to its non-invasive and undamaged probe ability.However,the resolution of perfect conventional microscopy is limited into 200 nm due to the wave length used in the optical microscopy.Because of the Abbe diffraction limitation,the resolution cannot be improved further,in result,the Biology information is masked under 200 nm.Countless researchers proposal plenty of means to obtain the information under the diffraction limitation.Not only the scanning tunnel microscopy,the electron microscopy,the atomic force microscopy but the scanning near field optical microscopy are all the approaches to acquire the microcosmic information in different fields.However,these image approaches have the problems in imaging the thick inner information and the living cells samples.Research urgent needs a high resolution microscopy which can obtain the inner sample information non-invasively.Superresolution is a good technology which solve this problem.Recently,there are three kind of well developed super-resolution technology,stimulated emission depletion(STED)microscopy,structured illumination microscopy(SIM),single molecular localization microscopy(PALM\STORM).As one of the super-resolution microscopy,STED microscopy is the only microscopy which can obtain the high resolution in the scale of physics.And it has the advantage because this kind of microscope is based on conventional confocal.This thesis started with the building of STED super-resolution microscope,carried out a serious of research on the building and application of STED microscopy.1)The building of pulsed laser STED.This thesis filters two wavelength from the supercontinuum laser,one of them act as the excitation laser,the other act as depletion laser.In the progress of building the microscope,this thesis solve a lot of engineering problems,such as optics fiber lunch system,spatial light filter system,laser pulse synchronization,4f system.At last,we obtain the size of the fluorescence sphere approximate to its real size.And we obtain 80 nm resolution on the biology sample(microtubule system)and we realize the DNA origami superresolution image.2)Continuous wave STED.In contrast to the pulsed laser,continuous wave don’t need pulse synchronization leads to its low cost and maintenance.We realized several fluorophores STED super-resolution STED microscopy,as well as the GFP,p-dots,and we obtained 70 nm resolution on biology sample.In addition,based on the existing theory,this thesis simulates the 3D-STED Z PSF.Based on the results of simulation,we obtained the light spots in Z axis.This result means that we have the ability on building the 3D STED microscope.3)FRET assistant STED microscopy theory simulation.This paper proposal a strategy to improve the resolution of STED based on the STED microscope.We used MATLAB write a program to simulate the different condition in FRET assistant STED microscopy,including apply the depletion laser on the acceptor,apply the depletion laser on the donor,apply the depletion laser on the donor and the acceptor simultaneously.In the end we obtained the results that the best performance FRET assistant STED is applying the depletion laser on the donor and acceptor simultaneously.Based on this simulation,we design a high FRET efficiency pair,and we use this FRET pair to carry out the experiment to determine that the simulation is right.4)STED multicolor experiment.It is not suit for STED to carry out the multicolor experiment for the reason that STED need two lasers,and the excitation laser need to apply on the approximate maximum excitation spectrum,at the same time the depletion laser need to apply on the tail of the emission spectrum.So this thesis proposal a new strategy to base on step by step in field staining.We write a selfcorrelation algorithm program to overlay the different targets,and to solve the problem that the sample can be used only once,we acquire different areas in one field of view.In the end we obtained dual color super-resolution on microtubule and mitochondria.5)Photobleaching microscopy.Until now the super-resolution microscopy technology need the modification of the hard ware or software.In this thesis,we proposed a brand new super-resolution approach based on the photobleaching.This microscope only need a conventional confocal microscope,however,the excitation laser is much higher than the conventional confocal microscope.This paper investigate the pbotobleaching microscope from simulation model to nanosphere determination.In the end we obtained several fluorophore photobleaching super-resolution on biology sample.