In this application, we propose to purchase a new Confocal Microscope in order to meet the needs for more advanced and versatile imaging tools by VA funded research programs including fixed imaging of a multitude of targets simultaneously, high-resolution discrimination of subcellular localization, and live cell imaging over hours to days. This was sought in a single instrument, finding that the Nikon A1R can accommodate all these challenges. This system is both a replacement and improvement of the Pittsburgh VA R&D service capabilities. The current Confocal microscope (Olympus FluoView(tm) FV1000 Confocal Microscope) is 10 years old and lacks the capabilities illustrated above necessary to meet the needs for imaging fixed and live cells in current and future research projects. The Medical Center has committed matching funds to absorb 25% of the cost of the integrated microscope system. To accommodate the multiple simultaneous detections, we have selected a laser launch with 6 laser lines (405, 440, 488, 514, 561, 640nm) that allows for more flexibility in wavelength selection and the use of multiple antibodies to image cellular structures. There are 12 VA investigators in this grant with projects that range from neurological conditions, such as strokes, brain tumors, traumatic brain injuries, motor neuron degeneration, lung diseases, sepsis, fungal infections, bone physiology, various types of cancer and metastatic disease. This capability is needed by every investigator's project in this application. To increase XZY resolution we have selected the Super Resolution function N-SIM package that affords imaging beyond the diffraction limit with 2x improvement in traditional optical resolution limits and enhances imaging resolution. This function is critical for several projects focused on imaging osteoblast matrix vesicles, synaptic microdomains in which specific proteins may exert effects on psychosis risk in dementia, interactions of ion channels and growth factor receptors and protein bridging the cytoskeleton to the membrane in cancer cells. Total Internal Reflection Fluorescence microscopy (TIRF) is designed for imaging endo/exocytosis, protein trafficking, cell-cell interactions, focal adhesions, and cell-matrix distances. TIRF is critical for the imaging needs of VA funded research focused on a broad spectrum of disorders, such as dementia by imaging dendritic spine complexity, ion transporters in osteoblasts and osteoclasts, changes in fungal membranes in response to antifungal agents, growth factor receptor and ion channel interaction in squamous cell carcinoma and cytoskeletal linkage to membranes in tumor cell migration. The microscope will be equipped with a stage top environmental control system for live cell imaging applications, which maintains environment in order to image cells over time. Projects that use in vivo cell imaging for up to 7 days are focused on osteoblast motility and cell density in vivo, changes in tumor cell phenotype, interactions between chloride channel and growth factor receptors in carcinoma cells and fungi live cells that are treated with antifungal agents. Nikon's Perfect Focus System eliminates focal drift and combined motorized stage allows to successfully image multiple fields of view / cells over time. Finally, wide field epifluorescence and bright field imaging capabilities are included and needed for each of the projects. The acquisition of this equipment is timely with the opening (expected by the end of summer 2016) of the new VA Pittsburgh laboratory facilities, where this equipment will be housed. The overall impact of the acquisition of this equipment is broad considering that 12 established VA investigators will be regular users of the microscope and additional PIs are expected to become users in the future as part of a growing cadre of new VA funded scientists. In summary, this equipment represents a long-term investment aimed at retaining funded VA investigators and attracting new VA investigators with the ultimate goal of increasing the pace of scientific and translational discoveries that will lead to new treatments for Veterans.
Research at the VA Pittsburgh encompasses a broad spectrum of medical fields and is aimed at improving diagnosis and treatment of a wide range of disorders that are highly prevalent in Veterans, as well as in the general population, and that represent a priority for ORD funding and strategic planning. Major areas covered by research at the VA are neurological disorders, pulmonary diseases, cancer, infectious diseases, sepsis and bone physiology. All research projects use of cell and organ culture systems that require imaging-based analyses. The overall impact of the acquisition of the confocal microscope is broad considering that 12 established VA investigators will be regular users of the microscope and additional PIs are expected to become users in the future. This equipment represents a long-term investment aimed at enhancing the research capabilities needed for VA funded research with the ultimate goal of increasing the pace of scientific and translational discoveries that will lead to new treatments for Veterans.
