Advanced two-photon fluorescence microscopy techniques for preclinical brain research

Two-photon fluorescence microscopy is an indispensable tool in modern systems neuroscience, with its high spatial resolution, high temporal resolution, and ability to harness the target specificity of genetic tools. Its usefulness is underscored by its ubiquity in both technology-driven and science-driven research laboratories around the world, made even more accessible with off-the-shelf microscopes sold by many industry partners. Although incompatible with human clinical research due to a need for an exogenous fluorescent indicator, two-photon fluorescence microscopy shines when applied to preclinical small animal models such as the laboratory mouse that can harness a full library of transgenic and acute genetic tools to study healthy and diseased physiology. Insights learned from imaging experiments performed in these animal models can then be used to guide clinical therapies in human patients. This thesis aims to develop and validate novel neural technologies that marry two-photon fluorescence microscopy with preclinical research applications, while keeping accessibility as a core goal./nThe first section of this thesis explores the use of Bessel two-photon microscopy in the study of neurovascular dynamics. Characterizing the dynamics of vasodilation, vasoconstriction, and blood flow in the brain can guide therapies for neurovascular pathologies such as traumatic brain injury and stroke. This study establishes Bessel two-photon fluorescence microscopy as a superior method for observing neurovascular dynamics over the conventional Gaussian two-photon fluorescence microscopy due to its fast volumetric imaging speed and integration of fluorescence signal. An embodiment of accessible technologies and in collaboration with an industrial partner, a commercial Bessel two-photon fluorescence microscopy system was designed, built, and used in this study./nThe next section of this thesis introduces and implements two techniques that complement Bessel two-photon fluorescence microscopy: adaptive optics and Bessel-droplet focus. Both methods improved the imaging quality of a commercial Bessel two-photon microscope without the need of additional hardware./nThe final section of this thesis combines two-photon fluorescence microscopy with cortical electrical stimulation to understand how the cortical microcircuit responds to electrical perturbation. Cortical electrical stimulation can be a powerful tool in clinical treatment, but its application has been hampered by a lack of understanding on the input-output relationship between stimulation parameters and neural response, as well as how stimulation can perturb regular cortical functions such as sensory processing. By performing concurrent brain-surface electrical stimulation and two-photon imaging of genetically defined neuronal subtypes in the awake mouse, this study uncovers a mechanism where the sensory-evoked excitatory cortical response is reduced through direct electrical activation of an inhibitory neuron population./nThe technologies and discoveries described in this thesis, through their accessibility and clinical relevance, aspire to enable future research pursuits of scientific discovery and therapy development.

Advisor: Ji, Na