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 hexa-pod piezo-electric positioning stage. This stage has been completely integrated into the operation of the microscope. Motion compensation algorithms have been also developed to compensate for in-plane displacements resulting in higher signal to noise performance. Using these approaches studies on the physiological effects of exercise, hypoxia and various genetic manipulations are being initiated. 2) Using TPEFM we have defined the macromolecular structure of the arterial wall of the porcine coronary and murine aorta. These data reveal, for the first time, the full 3 dimensional microstructure of collagen and elastin in these structures. We discovered that the uniquely exposed polyglycans located at the vessel branch points specifically bind LDL. This binding is highly cooperative, initially reflecting an electrostatic interaction with the macromolecules followed by a hydrophobic self association, resulting in the cooperative behavior. The steady state kinetics of LDL binding to vessel walls reveal an inflection point at approximately 150 mg/DL, close to the plasma value where non-linear impact of changes in cholesterol are reflected in non-linear clinical outcomes. The highly cooperative nature of LDL binding to macromolecules could be partially responsive for this phenomenon. We are currently using this approach to map the entire macromolecular structure of the renal artery, a highly susceptible vessel to atherosclerosis. 3) 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) uses a mirror system that redirect all of the emitted light to separate photomultiplier tube during the TPEFM process increasing the signal to noise of the experiment by over a factor 5 and the time efficiency by a factor of 25. Clearly, this approach is currently the most efficient method of imaging any fluorescent probe.
