Adaptive Optics and structured illumination in fluorescence microscopy
Thomas, Benjamin Alan
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Fluorescence microscopy has become one of the most important tools for biological investigation. Unfortunately, diffraction limits the resolution of fluorescence microscopy to about 250nm for high numerical apertures, leaving many cellular features unresolved. Several techniques have been developed to breach the resolution limit of fluorescence microscopes, one of which is structured illumination microscopy (SIM). SIM is based on the Moiré effect, where the interference of two patterns creates a lower frequency “beat” pattern. In SIM, the sample is illuminated structured pattern. The super-resolution details are encoded into the observed image, but can be mathematically extracted and restored to their proper positions. However, super-resolution details cannot be accurately restored if the illumination pattern is aberrated, which occurs as it passes through a complex biological sample. SIM is thus limited to thin samples without some means of correction, making in vivo imaging impossible and limiting its usefulness Adaptive Optics (AO) offers a means for correcting sample aberrations, restoring image and illumination pattern fidelity. Over the past 20 years, AO has been applied to various forms of microscopy. AO systems work by removing aberrations in the optical system through a correction element, which is typically a mirror whose shape can be precisely controlled. In this dissertation I will present my contributions to the fields of SIM and AO during my graduate career under the direction of Dr. Peter Kner. I will present a new form of optical sectioning SIM and demonstrate its superior performance in comparison to previously reported methods. I will also present our application of AO to Differential Interference Contrast (DIC) microscopy, which has not yet to be reported in literature. This is important as it offers a means of aberration removal for photosensitive samples. Most importantly, I will detail the combination of AO and SIM and demonstrate that 140nm resolution images can be obtained through 35μm of sample tissue.