Two Photon Adaptive Optics Fluorescence Lifetime Imaging Ophthalmoscopy PDF Download

"The retina is the light-sensitive, multilayered tissue at the back of the eye responsible for converting light into electrical impulses for visual perception. Dysfunction in even one cell type or layer can result in partial to total blindness. Observing the structural and functional dynamics that underlie dysfunction, especially before cell death, is critical to understanding retinal diseases, developing new diagnostic metrics, and evaluating novel treatments. Adaptive optics scanning light phthalmoscopy (AOSLO), which permits near-diffraction limited imaging by correcting the inherent aberrations of the eye, has enabled in vivo subcellular scale evaluations of the retina. Two-photon excited fluorescence (TPEF) imaging allows the optical probing of molecules spectrally inaccessible with single-photon fluorescence that play important roles in metabolism, the visual cycle, and structure. By combining TPEF with AOSLO, it may be possible to evaluate the biochemistry of different cells and layers throughout the living retina and relate those measurements to function. Previous work has established the instrumentation and workflow to perform TPEF adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO), an AOSLO modality which measures the time-dependent component of fluorescence aggregated from all contributing endogenous and exogenous fluorophores. The goals of this thesis are to determine how lifetime signatures differ between cells and layers and disambiguate the aggregate lifetimes into their constituent molecular species. First, AOFLIO was deployed in macaque photoreceptors. The phasor method of analysis, a method to visualize fluorescence lifetime decays in two-dimensional frequency space, was incorporated into this workflow. This enabled the separation of S cone, M/L cone, and rod photoreceptor lifetime signatures, an improvement in sensitivity over traditional multiexponential fitting. Second, AOFLIO and phasor analysis were applied to other features in the macaque retina. In vivo fluorescence signatures can be compared to those of known retinal fluorophores with a phasor fingerprint, allowing inferences about the dominant contributing sources. Finally, AOFLIO was deployed in rho-/-retinitis pigmentosa (RP) mice whose retinas expressed a fluorescence lifetime-based sensor of glucose concentration. The first evidence of glucose sequestration in the retinal pigment epithelium, the hypothesized mechanism that causes sequential cone death in RP, was observed. This work has advanced the development of TPEF AOFLIO as a noninvasive probe of biochemical composition in the in vivo retina. This may allow subcellular evaluations of the retina in health, throughout the time course of disease, and in response to therapeutics"--Pages xviii-xix.