The purpose of these studies was to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. The major approach used was non-linear optical microscopy techniques. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have completed a next generation system for motion correction that performs a complete 3 dimensional correction scheme in near real time using a resonant scanning microscope and a graphical processing unit (GPU) to rapidly process the data. This permits the 3 dimensional correction for motion within the biological tissues in vivo. This novel interface of a true real time 3D imaging technique with a GPU provides the first real time motion correction scheme for intra-vital microscopy. 2) We have adapated this technology to generate large field of view (centimeters)images with micron in plane resolution. This provides a novel view of the overall microvascular and cellular structure of tissues previously not available. This approach has been applied to the brain, heart, liver and skeletal muscle. 3) We have completed our preliminary studies on the application of adaptive optics to correct for the distortion of the excitation light in these studies. Using this approach we have been able to greatly improve the penetration and signal to noise of these measurements. Full implementation of this system into our standard scope is now underway. 4) Using the inherent nature of TPEFM we have completed the development of an imaging scheme that collects nearly all of the emitted light from a probe during the imaging experiment. This approach termed Total Emission Detection (TED). We have currently modified this initial concept to include a surface collecting scheme compatible with in vivo measurements (so called epi-TED). This system has shown to improve the signal collection from fluorescence imaging experiments in vivo by a factor of 2-4 fold. Clearly, this approach is currently the most efficient method of imaging any fluorescent probe in vitro or in vivo.
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