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Super-resolution Imaging of Biological Samples using Linear and Nonlinear Structured-Illumination Microscopy


Structured-Illumination Microscopy (SIM) can increase the spatial resolution of a wide-field light microscope by a factor of two with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. In this dissertation, I apply linear SIM to biological samples in both two and three dimensions and use ultra-low light intensities that are well suited for investigating biological samples to demonstrate whole-cell super-resolution imaging by Nonlinear Structured-Illumination Microscopy (NL-SIM). Previously, NL-SIM has achieved ~50-nm resolution on dye-filled polystyrene beads by saturating the excited state of a fluorophore [Gustafsson M (2005) Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102(37):13081-13086]. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders-of-magnitude lower than those needed for saturation. We experimentally demonstrate ~40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, NL-SIM is now a biologically compatible super-resolution imaging method.

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