Multiphoton microscopy is a technique that involves the imaging of fluorescently tagged cells within intact tissue using laser scanning microscopes and pulsed infra-red lasers. This approach can be employed with either 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 their research would allow for rapid progress and provide substantial insights into their in vivo biological questions in 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 scanning microscope (LSM) and multiphoton (MP) laser. Yale University School of Medicine investigators are fortunate in that the school has already invested in the multiphoton laser and has purchased an additional custom designed laser scanning microsocpe equipped with sensitive external detectors that allow for the collection of low level light. A unique microscopy suite is now under construction that will permit the preparation of animals and their subsequent imaging to occur in adjacent rooms. This microscopy system will be available to Yale investigators, including those members of the YRDRCC. Nevertheless, many barriers currently exist that prevent investigators from utilizing this approach to their in vivo research. For example, 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 the animal anesthesia, surgery and restraint during IVM appear unobtainable to most researchers, given that relatively few investigators within immunology have employed IVM in their research and are thus available for training and advice to the broader immunology community. Likewise, the detection of fluorescence in vivo often requires strains of mice expressing fluorescent proteins that can be difficult to obtain in a timely fashion. Therefore, this application proposes to establish an In Vivo Imagng Core Facility that would not only reduce 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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