Optimization of two-photon excited fluorescence for volumetric imaging

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain.