IMPROVING LONG-TERM, LIVE-CELL FLUORESCENCE MICROSCOPY

Fluorescence microscopy is one of the most powerful tools for studying sub-cellular dynamics with molecular specificity at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of a detection objective using an orthogonal excitation path with a laterally restricted illumination pattern. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both the detection efficiency and native spatial resolution of the detection path. To address this limitation, we present a novel live-cell LSFM method: Lateral Interference Tilted Excitation (LITE) microscopy, in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, efficient, coverslip-based detection objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. Additionally, we formulate and test an optical model to explain the photobleaching improvement that we observe when imaging thin (~5 μm) fluorescent organisms.In addition to developing microscopy technology that improves photobleaching over conventional fluorescence microscopy, we also have proposed theoretical optics to further remove imaging limitations for live-cell biology. Conventional optical microscopy relies on the refraction of light with lenses to magnify and resolve specimens; however, comparatively little work has been done on using reflection as a means of microscopy. We therefore propose using paraboloidal mirrors as primary illumination and detection optics to improve illumination consistency and detection efficiency in fluorescence microscopy.Doctor of Philosoph