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Fluorescence based adaptive optics and multidimensional fluorescence microscopy

Creative Commons 'BY-NC' version 4.0 license

Studying the structure and interactions of molecules and cells in their native environments has always been a challenge in the life sciences. When an organism of interest is physically out of reach or invisible to the naked eye, scientists have historically turned to reductionist experiments that first isolate the organism from a complex environment and then study the bulk signal from a large population of cells in tissue or in bacterial colonies. However, the structure, function, and interactions of a population of cells can vary at a subpopulation and even single-cell level. Non-invasive tools that can study a population in relevant conditions, such as in tissue, capable of resolving single cells in a large region of interest are necessary to improve treatment of disease and understanding of physiological phenomena.

Microscopy – in particular multiphoton fluorescence microscopy – holds enormous potential to discover new science by elucidating biological properties with subcellular resolution. When paired with adaptive optics, multiphoton microscopy can image deep into highly scattering tissue, resolving structures at previously inaccessible depths. The biochemical composition of a sample can be determined with fluorescence lifetime microscopy and hyperspectral microscopy, giving information about cell state and metabolism. Image correlation spectroscopy and single molecule tracking methods can yield information about the dynamic sample properties such as transport mechanisms and diffusion of genetically encoded fluorescent proteins.

The work presented in this thesis is separated into three chapters. The first chapter describes the development of a novel deep tissue multiphoton microscope, the AO DIVER, which uses a network of guide stars to perform millimeter-scale 3D adaptive optics imaging and enable millimeter-scale imaging. In the second chapter, the deformable mirror used in the AO DIVER is used to create a high speed 3D scanning instrument, capable of scanning axially and laterally at rates of up to 40 kHz. Finally, in the third chapter, a program for simultaneous unmixing of hyperspectral and lifetime fluorescence images is described and implemented in Pseudomonas aeruginosa biofilms, elucidating a depth dependent physiological change in bacterial biofilms.

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