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 continued to make improvements in the technology of this approach by expanding our rapid z-focusing system with a full X-Y-Z motion correction scheme using a new resonant TPEFM system from Leica providing near real time 3 dimension data with a graphical processing unit (GPU) to perform near real time 3D motion correction of 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) Using an earlier version of this motion compensation system we have been successful in determining the 3D structure of the cells and vascular structures in several organs including the kidney, skeletal muscle and liver, in vivo. These structural studies have provided unparalleled views of this tissue microstructure providing insight into how the microcirculation is coupled to the functional cellular elements. 3) We also applied this technology to monitor the intracellular metabolic responses of skeletal muscle mitochondria to global hypoxia, in vivo. We determined that different pools of mitochondria within the cell are poised at different redox states in the resting muscle. The mitochondria located adjacent to capillaries was found to the significantly more oxidized than mitochondria deep inside the cell in the so called intrafibrillar regions. In addition, we demonstrated that the mitochondria are concentrated in the paravascular regions compared to the intrafibrillar regions. These observations led to the novel hypothesis that the distribution of mitochondria is contributing to an oxygen gradient across the cell, high near the vascular structures and very low in the remaining, majority of the cell resulting in an overall low cellular PO2 at rest. This maintenance of a low cellular PO2 may be advantageous to prevent the generation of reactive oxygen species or protein oxidation when the muscle is at rest. Direct measurements of the oxygen tension in the cell are currently being attempted. 4) Using the inherent nature of TPEFM we have developed 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. 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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