Superresolution Microscopies Based On Several Nonlinear Effect

Optical microscopy is a necessary tool for biologists to research the structure anddynamics of cells noninvasively, and the development of microscopy accompaniedwith the advancement of life science. However the presence of diffraction limited theresolution of microscopy to above200nm, which hindered the further research ofbiologists. Fortunately, the emerging of superresolution microscopy technique hasbroken this limit and offered a powerful tool for biologists. This dissertation presentsseveral techniques of superresolution imaging that based on nolinear effect offluorescence for providing higher resolution and simpler imaging method. The mainresults are as follows:(1) We built a stimulated emission depletion microscopy (STED) based on acommercial laser scan confocal microscopy. By imaging100nm fluorescent beads,cellar microtubule and DNA origami with STED, we find that both the resolution ofSTED is better than100nm。We observed the Lamp1protein and clathrin in Hela cellswith confocal and STED respectively. The imaging results showed that the resolutionand contrast of STED is obviously better than confocal and the STED could be usedto image cellular samples.（2）We proposed a superresolution technique by combing the Fluorescenceresonance energy transfer (FRET) and STED. The resolution could be enhanced eventhough the peak of emission wavelength of donor is far from the depletion wavelength.The theory model was set up and experiment results were in accordance withsimulated results.(3) We proposed the resolution of FRET microscopy can be improved by timegated detection. The lifetime of FRET donor varies with the excitation for thesaturation of FRET accepter. With time gating technique, the temporal dynamics ofthe spontaneous emission of the donor molecule could be used to encode the spatialinformation and enhance the resolution of microscopy three dimensionally with thehelp of time-gating. As the lifetime of fluorophores can also be changed by the STED,we applied a depletion beam to accepter to modulate the lifetime of accepter andtherefore modulate the lifetime of donor. The simulation shows that the resolution isunlimited even with limited depletion intensity. (4) We developed a high FRET efficiency system which was based onnanobodies and fluorescent proteins. The FRET occurred between fluorescent proteinand organic dyes that covalently coupled on the nanobody. Benefiting from the smallsize of nanobody, the FRET efficiency could reach up to97%. We validated thisconcept in fluorescence spectroscopy and confocal laser scanning microscopy. TheFRET imaging data of varies of cellular organs and fluorescent proteins showedimpressive robustness and versatility of this system. The high efficiency FRET systemcould be used in superresolution microscopy that based on FRET.(5) An easy superresolution technique based on photobleaching was proposed.The superresolution imaging could be performed in confocal microscopy. Firstly weintroduced the theory and carried out simulation with MATLAB. Then severaldifferent kinds of fluorescent beads were used in photobleaching experiments. Theresults showed~60nm resolution and compared with confocal imaging the resolutionwas enhanced3-4times. We performed photobleaching superresolution imaging ofcellular microtubule and obtained~100nm resolution. We colabeled the fluorescentbeads and microtubule with both Alexa405and Atto488respectively and performedphotobleaching imaging with different wavelength excitation lasers. The resultsshowed that this photobleaching superresolution technique can realize dual colorsuperresolution imaging.