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Biological fluorescence imaging on the nanoscale [electronic resource] : STED super-resolution microscopy of cells and tissues

A majority of biological microscopy investigations involve the focusing of visible light with conventional lenses. Fluorescence microscopy is one of the most widely used tools in biology but its resolution has historically suffered from the diffraction limit to about 200 nm laterally and 800 nm axially. In the past decade, this resolution problem has been overcome by the rapidly emerging field of super-resolution microscopy. The first demonstrated super-resolution technique, STimulated Emission Depletion (STED) Microscopy, is the topic of this Dissertation. This Dissertation has two primary areas of focus: the design optimization of a STED microscope, covered in Chapters 2-4, and its application to super-resolution imaging in cells and tissues, covered in Chapters 4-6. Chapter 2 describes the STED apparatus and experimental methods used. This chapter covers the guiding principles behind the design of a STED microscope, which forms a basis for understanding the logic underlying the homebuilt STED microscope which was constructed for this research. This STED microscope has a typical resolution of approximately 60 nm (full-width-at-half-maximum) or 25 nm (sigma) and has the sensitivity to image single fluorophores. In Chapter 3, a framework for evaluating and optimizing STED performance in the presence of several key tradeoffs is presented. Chapter 4 describes both developments in STED Microscopy required to utilize far-red-emitting dyes and the challenges associated with performing super-resolution imaging in intact Drosophila tissue. In Chapter 5, the optimization of labeling density revealed the 9-fold symmetry of a centriole protein structure, an important organelle in cell development. In Chapter 6, Huntingtin protein aggregates are resolved beyond the diffraction limit in a cell model of the neurodegenerative Huntington's disease.