This application requests funds for purchasing an imaging workstation that enables stereological measurements based on multichannel fluorescence identification of features to be counted, time-lapse live cell fluorescence imaging and measuring Fluorescence Resonance Energy Transfer (FRET) between cyan and yellow fluorescent proteins (CFP and YFP). Morphometric measurements, such as numbers and sizes of cells, density of neuronal fibers in brain circuits, density of synapses in specific areas or on specific cells, number and stages of ovarian follicles are important determinants of physiological changes associated with conditions that constitute the focus of research of multiple NIH-funded grants at the Oregon National Primate Research Center (ONPRC). Estimating these parameters based on two-dimensional microscopic images is very laborious and is affected by biases, if sampling and analysis techniques of modern stereology are not followed. A stereology compatible imaging system is therefore necessary for the research of our investigators. For other projects, use of green fluorescent proteins and their mutants has created opportunities for identifying the intracellular location, translocation and function of genes of interest. A particularly attractive application is the visualization and measurement of the interaction between biomolecules in physiologically relevant states, in real time, using FRET, especially between CFP and YFP. This is not possible with the instruments currently available to us, but necessary for our NIH-funded investigators. This workstation will be part of the Imaging and Morphology Core of ONPRC located on the West campus of the Oregon Health & Sciences University and will enable the core to provide services that are either not possible now, or that will be significantly improved in accuracy and speed. An internal advisory committee will establish procedures and oversee the general operation of the imaging workstation. The principal investigator will be in charge of the installation, training, scheduling, use and maintenance of the new imaging workstation. The institution is committed to maintenance of instrument and providing oversight, support for staff salary and training.
