Research On Super-resolution Microscopy And Instrument Based On Modulation Of Fluorescence In Time And Space

Far-field optical microscopy has long been a great tool in biomedical researches for its non-invasive, highly specific, deep penetration depth properties. However, due to the existence of the optical diffraction limit, the resolution of far-field optical microscopy has been restricted to about 200 nm, which cannot meet the increasing demands of modern biomedical researches. Confocal microscopy improves the resolution of far-field optical microscopy effectively, but its resolution is still limited by the optical diffraction limit.Fluorescence microscopy has many advantages such as high signal-to-noise ratio, high specificity and multiple-parameters, which makes it a perfect match for long-term observation of biological samples. "Selective labeling, selective excitation", the imaging characteristics of fluorescence microscopy, can break the diffraction limit in theory. Therefore, super-resolved techniques based on fluorescence theory are the most important representatives of far-field optical super resolution microscopy. However, there still exist many problems in today’s super-resolution technologies, such as the low resolution, the complex system, the narrow applicability scope of samples, slow imaging speed, etc.Based on fluorescence theory and confocal techniques, the thesis is meant to get higher resolution, simplify the system, enlarge the applicability scope of samples, increase the diversity of imaging methods, and finally realize the super-resolution imaging of fluorescence samples at sub-100nm resolution. The main points are as follows:(1) Two methods and systems based on modulation of the detection point spread function were put forward:By simply modification of the confocal system and "virtual modulation" of the fluorescence, super-resolution methods based on modulation of the detection point spread function can achieve a simple super-resolution system with high resolution, real-time imaging, high signal-to-noise ratio, and a wide applicability scope of samples.(2) Based on time-gating technology and fluorescence modulation by stimulated radiation in time and space, an off-line time-gated STED super-resolution system was put forward. On the other hand, a novel aligning method base on fluorescence lifetime distribution was put forward. A resolution of about 38nm-50nm can be achieved by our system. The system has been greatly simplified compared to ordinary STED, and the intensity of the stimulated radiation beam has been greatly reduced.(3) Dual-mode super-resolution microscopy based on STED and FED was designed and built:By combining STED and FED two super-resolution modes in one set, this system has properties such as a wide applicability scope of samples, a fast scanning speed and a high resolution, etc. A resolution of 110 nm can be achieved by the STED mode, and a resolution of around 140 nm can be achieved by the FED mode.(4) Preparation methods for the anti-bleaching mounting medium, beads samples and biological samples were studied. The study has important significance in solving the bleaching issue in STED microscopy.(5) Sub-diffraction imaging method based on stochastic bleaching and single molecule localization techniques were proposed. To solve the bleaching problems, the best way is to use it instead. This method only uses an ordinary confocal microscope, which means high application significance. This method has advantages such as high resolution, a wide applicability scope of samples, a simple system, etc.