Multiphoton microscopy is a technique that involves the imaging of fluorescently tagged cells within intacttissue using laser scanning microscopes and pulsed infra-red lasers. This approach can be employed witheither excised bulk tissue or with live, anesthetized animals, a technique referred to as intravital microscopy(IVM). Many investigators within the proposed YRDRCC feel that the application of these techniques to theirresearch would allow for rapid progress and provide substantial insights into their in vivo biological questionsin a way that is not possible with traditional in vitro assays. However, in order to perform this technique,equipment is required that is prohibitively expensive to all but a few investigators, namely a laser scanningmicroscope (LSM) and multiphoton (MP) laser. Yale University School of Medicine investigators arefortunate in that the school has already invested in the multiphoton laser and has purchased an additionalcustom designed laser scanning microsocpe equipped with sensitive external detectors that allow for thecollection of low level light. A unique microscopy suite is now under construction that will permit thepreparation of animals and their subsequent imaging to occur in adjacent rooms. This microscopy systemwill be available to Yale investigators, including those members of the YRDRCC. Nevertheless, manybarriers currently exist that prevent investigators from utilizing this approach to their in vivo research. Forexample, the cost to users of the MP laser is significantly higher than the cost of the microscope by itself,largely due to the service contracts for maintenance of the MP laser. The additional skills needed for theanimal anesthesia, surgery and restraint during IVM appear unobtainable to most researchers, given thatrelatively few investigators within immunology have employed IVM in their research and are thus availablefor training and advice to the broader immunology community. Likewise, the detection of fluorescence in vivooften requires strains of mice expressing fluorescent proteins that can be difficult to obtain in a timelyfashion. Therefore, this application proposes to establish an In Vivo Imagng Core Facility that would not onlyreduce these barriers but also provide training and advice in the imaging of deep tissue and live animals.The proposed IVICF would have a dramatic impact on the pace of research into cell-cell interactions,differentiation and migration in vivo in both health and in rheumatic and autoimmune diseases.
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