Two-photon laser scanning microscopy is a technique that can be applied to the imaging of fluorescently tagged cells within intact tissue using point 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). In order to perform this technique, equipment is required that is prohibitively expensive to all but a few investigators. Yale University School of Medicine investigators are fortunate in that the school has already invested in a two-photon laser and custom designed laser scanning microscope equipped with sensitive external detectors that allow for the collection of low level light. A unique microscopy suite for use by this Core C permits the preparation of animals and their subsequent imaging to occur in adjacent rooms. This facility has been subsidized through a P30 Core grant and made available to all members of the YRDRCC. For a multitude of these investigators, the application of this techniques to their research has greatly accelerated the progress of their research and provided substantial insights into their in vivo biological questions in a way that is not possible with traditional in vitro assays. Together with the Core PI, the facility operator has consulted with investigators to ensure that experimentation came to fruition quickly and efficiently. P30 funding has made possible substantial methods development that enabled many novel applications, and expanded the fluorophore combinations and tissue types that could be imaged. Nevertheless, without additional P30 Core funding, many barriers that prevent investigators from applying this approach to their in vivo research would resurface. For example, the cost of the service contracts for maintenance of the laser and microscope would require that facility user fees be greatly increased, posing a significant financial deterrent that would prohibit its use by many investigators, especially junior PIs. The additional skills needed for the animal anesthesia, surgery and restraint during IVM appear daunting to most researchers, despite the training and advice that we have made available. Therefore, this application proposes to expand the capabilities and further promote the In Vivo Imaging Core Facility. P30 funding would not only reduce these barriers but also acquire additional methodologies to query as yet inaccessible biological events. The proposed IVICF would have a dramatic impact on the pace of research into cell-interaction, differentiation and migration in vivo in both health and in rheumatic diseases.
This application proposes to expand the capabilities and further promote an In Vivo Imaging Core Facility that allows fluorescently tagged cells to be imaged within living anesthetized mice. P30 funding would reduce numerous significant barriers and allow for the development of additional methodologies. The proposed core would have a dramatic impact on the pace of research in both health and in rheumatic diseases.
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