The purpose of these studies is 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 further modified our motion tracking schemes to intermittently track tissue between other measures of either low intensity or light sensitive process or physiological stimulation such as muscle contraction, where the constant illumination or tracking of the sample is not desirable or not feasible. This added complexity to the tracking scheme has permitted monitoring photoactive fluorescence proteins as well as initialize studies using muscle contraction as a physiological perturbation, in vivo. The programming of this system now permits complete control of numerous laser excitation schemes, physiological control as well as detection paths in real time. Thus, the microscope is being converted into a physiological monitoring device rather than a simple image collection system. Our primary targets in this program are: tracking mitochondrial motion and fusion in vivo, monitoring intracellular events associated with muscle contraction, in vivo and monitoring cell division in a liver model of cellular regeneration. 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. Applying this technology to the skeletal muscle in vivo, we have discovered that the capillaries associated with slow twitch muscle fibers are actually imbedded in the cells in highly specialized structures. This imbedding of the capillaries in the cells greatly improves the diffusional delivery of oxygen and substrates to the fiber but also dramatically restricts the delivery of these elements to that particular fiber and eliminated diffusional competition with other fibers in the region. Though these structures have anecdotally been decribed in the past, these full 3D volume studies reveal that these structures are the dominate delivery system for the slow twitch muscle fibers not appreciated previously. We are currently evaluating nature of the capillary grove in these cells attempting to establish the distribution of vascular element receptors and structural elements in the groove. 4) In conjunction with this study we are developing image processing schemes to extract the entire vascular bed from these 3D data sets to support modeling of blood delivery to the tissue. One interesting aspect of these studies is to predict the distribution and control of regional blood flow to different fiber types. In addition, we are also extracting information on the individual muscle fiber structures and mitochondria content to develop a more complete model of muscle architecture and regulation on the micron scale. 3) We have been continuing our studies on the use of adaptive optics to correct for the distortion of the excitation light in these studies. To date the improvements have been modest, however we continue to optimize the system to attempt to extract the true optimal performance in our in vivo environment.
Showing the most recent 10 out of 15 publications
