Multifocal multiphoton microscopy with adaptive optical correction

Multiphoton microscopy (MPM) is a remarkably versatile technique in biological imaging. MPM provides increased depth over confocal imaging and can be combined with other imaging techniques such as fluorescence lifetime imaging microscopy (FLIM), adding functional information. FLIM read-out is relatively straightforward using time-correlated single photon counting (TCSPC). Fluorescence lifetime detection enhances the power of multiphoton imaging to allow three dimensional, concentration independent, measurements of environmental parameters such as pH, Oxygen tension and Ca2+ in addtion to the interaction or conformational modification of proteins by Förster resonant energy transfer (FRET); the latter is a particular focus of the Dimbleby research groups at King’s College London. However, there are significant limitations in both FLIM and MM. Limitations of TCSPC-FLIM include prolonged acquisition times along with signal and resolution degradation as a function of depth. This thesis demonstrates advancements multiphoton fluorescence lifetime imaging through improvements in two principal areas: speed and resolution at depth. In order to improve acquistion rates a multifocal multiphoton microscope (MMM) capable of rapid, parallelized TCSPC-FLIM was developed-MegaFLI. Acquisitions demonstrate rapid 3-dimensional, high temporal resolution FLIM in vivo Zebrafish. Performed by massively parallel excitation/detection the speed is signficantly improved by a factor of 64. In parallel to the MegaFLI project, a second microscope employing adaptive optical correction has been developed. The introduction of Adaptive Optics (AO) serves to improve imaging quality by counteracting the refractive index heterogeneities introduced by the sample, limiting the imaging depth. Incorporated with a single beam scanning FLIM system, a pupil-­‐segmentation AO-TCSPC-FLIM demonstrates improved signal-to-noise ratio (SNR) and resolution, permiting a more accurate determination of fluorescent lifetime in turbid media.