This proposal requests funds to purchase a Molecular Dynamics 2001 Confocal Laser Scanning microscope. This instrument will serve the research needs of a number of faculty of Baylor College of Medicine whose research work focuses on the dynamics of living cells, the three- dimensional arrangement of subcellular structures, and the three- dimensional reconstruction of cells such as neurons in intact brain slices. We have a critical mass of investigators whose work involves microscopy of living cells extending from the molecular level to interactions between cells in tissues. Our investigators at BCM are conversant with the advantages and limitations of digital imaging fluorescence microscopy. The diversity in expertise includes optical microscopy, molecular biology, spectroscopy, image processing, analytical chemistry, microinjection, immunochemistry, cell biology, molecular graphics and structural and computational biology. In addition to the benefits of superior spatial resolution that are provided by multi wavelength confocal microscopy of the subjective interpretation of cellular phenomena, the applications of this technology envisioned by the BCM investigators have two other themes in common. The first is a need for information about the three-dimensional organization of subcellular structures and the time dependent change in their organization in response to specific perturbants. The second is a need to quantify cellular features and/or processes: dimensions, frequency of occurrence, rate of uptake or how an organelle moves -- attributes that can be represented by a number. Investigations extending from the molecular level to interactions between cells in tissues have reached the limits imposed by the subjectivity of older microscopic methods and by the difficulty in processing manually, sufficient information to create databases for analysis by statistical methods. Collectively, we have the experience and collaborations established to utilize state-of-the-art multiwavelength confocal instrumentation to collect and analyze new kinds of information using statistical criteria, thereby increasing the productivity and value of our ongoing research. Although our older microscopic methods and the more recent development of quantitative digital fluorescence imaging has served us well, we have reached a point where our research programs would progress faster if state-of-the- art instrumentation for optical microscopy were available. Over the past several years facilities devoted to light and electron microscopy have been developed that serve as a research resource for the Texas Medical Center, Rice University, and the University of Houston. Despite many established and productive collaborations, we need to take advantage of recent developments in optical microscopy. Images from thick preparations are difficult to obtain in digital format and the computational methods used to treat these are not satisfactory. Higher resolution images will be critical for the advancement and integration of our programs in microscopy. The confocal instrumentation requested here as part of the $100,000 matching funds from the Keck Center for Computational Biology represents an essential ingredient in our ultrastructural and digital microscopy center and would serve as a shared resource to the research community at Baylor, the University of Texas Medical School, Rice University, and the University of Houston.
