Study On Wide-Field Fluorescence Microscopy With Three Dimensional Nano-resolution

Visualization, tracking and analysing molecules and their events in cells are critical in understanding a complex biological system. So, as a tool being capable of visualizing the dynamic processes inside cells, optical microscopy has to meet the following requirements, namely molecule identification, nano-resolution, single molecule detection sensitivity, and temporal resolution as high as microseconds even picoseconds. The last two requirements are mainly determined by the detector used during experiment. At present, the first requirement is easy to be met for the fluorescence microscopy, because fluorescent labels can be specifically attached with the aimed molecules. As an optical imaging method, the spatial resolution of a fluorescence microscopy is limited by the diffraction. However, with the development of laser techniques, label material, label techniques, and weak signal detection techniques, more and more imaging methods are put forward and realized, and traditional resolution which is limited by the diffraction has been broken through by means of different tricks. After investigation and analysis of nano-resolution optical imaging methods existed nowadays, for the sake of application in live cell and its dynamic processes in the future, the thesis is focused on wide field imaging methods which are potential of high speed imaging compared with point scanning ones. The thesis will deal with structured illumination microscopy and single molecule localization microscopy. In the latter, the issue to resolve the problem of thick sample imaging with nano-resolution is mainly concerned. What have done in this thesis is listed as follows.(1) Structured illumination imaging based on Digital Micromirror Device (DMD)In this thesis, a programmable DMD instead of piezo activated grating was used to produce stable and reliable structured illumination. Status of micromirros was controlled by the software so that the structured illumation pattern on DMD could be displayed and phase-shifted. Improvement of sectioned ability and lateral resolution were realized by different algorithms. In sectioned imaging, an optical low-pass spatial filter was applied to block the diffraction order higher than±1st order. Because the illumination pattern was not in sinusoidal mode, the algorithm used here was discussed in detail. The results have shown that two sectioned images with different sectioning ability can be reconstructed from five raw images with different phases. The results have also indicated that defocused information can be filtered out effectively. The improvement of lateral resolution has been analysed and the images of biological samples with super-resolution have been acquired.(2) Single molecule localization microscopySeveral parameters, such as the number of detected photons generated by a single molecule, effective pixel size of the detector, and the background noise level, are critical in determination of centroidal localization of the interested molecule. So, under a optimized point spread function, the effect of above parameters on the accuracy of centroidal localization are simulated and analysed by statistic methods. Nano-resolution imaging based on single molecule localization is simulated with Matlab. Centroidal localization of an interested single molecule simulated by Monte Callo method is almost the same as determined by the analytical formula. Imaging of a sample consisting of several lines of molecules is also simulated. Results demonstrate that two lines of molecules separated by 20 nm can be resolved. Nano-resolution imaging has been realized onⅨ71 inverted fluorescent microscope with the spatial resolution (FWHM) of 48 nm where a single wavelength (640nm) laser is acted as a light source of activation, deactivation, and excitation simultaneously and images of filopodias with diameter of 78 nm have been reconstructed.(3) Axially selective activation by dual-wavelength incoherent interference illuminationResolution of a few tens of nanometers has been achieved in fluorescence microscopy with photoswitchable fluorescent molecules. However, for the thick samples, the background brought by the crosstalk of unwanted on-state fluorescent molecules is non-negligible, which results in a limited imaging thickness of the sample. So, even with inclined illumination, the largest thickness is not more than 3 microns. In this thesis, a background suppression method are presented, which is named as'selective activation of molecules within a thin layer in thick sample'. The core of our idea is dual-wavelength incoherent interference illumination, in which two axially modulated illumination distributions are generated by the interferences of two activation beams with the same phases and two deactivation beams with the opposite phases. Furthermore, the photoswitching characteristics of fluorescent molecules can be described by activation and deactivation rate, which are linearly dependent on the activation and deactivation laser power respectively, so the probability of molecules staying at activated state is also axially modulated, and at last the selective activation of molecules within a single thin layer is achieved. The performance of such a method is simulated with the known photoswitching characteristics of Cy5. With suitable parameters, the thickness of the thin activated layer can reach 35 nm (FWHM). For those molecules located out of the above central layer, the probability of molecules staying at activated state is depressed dramatically to be less than 6% of the central peak. Simulation results have demonstrated by this way that the background can be suppressed significantly for the sample as thick as 10μm.The main innovation in this thesis is that, a method named as "dual-wavelength incoherent interference illumination" is put forward to suppressing the background in single molecule localization microscopy, which is an obstacle when single molecule localization microscopy is applied for thick samples. Calculated results based on the known photoswitching characteristics of Cy5 have demonstrated that an activated layer as thin as 35nm can be acquired. Simulations results have also verified the perfect suppression of background. This method, combined with those axial localization methods, can be used in three dimensional nano-imaging of thick samples, such as a whole cell.