Enhancing the resolution of Single Molecule Localization microscopy using Quantum Dots and Adaptive Optics for imaging thicker samples
Forouhesh Tehrani, Kayvan
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More than a century after Abbe̕ discovered the diffraction-limit of a microscope; many new techniques were proposed and demonstrated which allow resolutions beyond the diffraction limit. These methods in general can be placed in two main categories; extension of optical transfer function (OTF), and optical shelving. The former category includes Structured Illumination microscopy (SIM), and the latter includes techniques such as Stimulated Emission Depletion (STED), and Single Molecule Localization (SML). In this dissertation we focus our attention to improving the resolution of SML; the accuracy of which is a function of several factors such as the brightness of the fluorophores, and sharpness of the Point Spread Function (PSF). Improvement of the photon emission of fluorophores have been addressed by the development chemical buffers that facilitate blinking with higher brightness, as well as brighter dyes such as Quantum Dots (QD). The QDs promise to improve the resolution by producing 17 times more photons than organic dyes, which dramatically increases the accuracy of the SML. The implementation of QD Blueing technique to SML provides a novel approach to overcome the high blinking duty cycle of the QDs. Here we present a novel two color QD blueing SML technique. Another critical challenge in improvement of the accuracy of the SML is by reducing the effect of optical aberrations. Aberrations tend to deviate propagating wavefronts from their flat form, and hence deform the shape of point spread function (PSF), which directly reduces the accuracy of SML. This can be caused by several factors including the imperfections in optical elements design, misalignments, and external factors such as refractive index mismatch in the propagating media. While the first two can be optimized by using aberration corrected elements and more effort on alignment, the latter cannot be reduced without the help of active optical elements. Biological samples induce aberrations due to their shape, and diversity of refractive index along their body. In this dissertation we demonstrate use of Adaptive Optics (AO) for compensation of wavefront aberrations for SML imaging on thick biological samples.