A number of investigators in the Departments of Anatomy and Cell Biology and Biochemistry at Georgetown University, School of Medicine, are requesting funds to purchase the newly developed Confocal Fluorescence Microscope. The uniqueness of this microscope system is its ability to optically section cells at different planes so that unwanted background fluorescent signals above or below the plane of section are excluded from view, thus providing a much more precise localization of antigens in specific regions of the cell. This novel technology is essential if one wishes to carry out immunofluorescence studies on cuboidal or columnar cells in culture without having to resort to sectioning. One can now optically section the cells at various depths and obtain fluorescence images of surprising lucidity. Many of the investigators in this proposal use extracellular matrix as substrate for their cultured cells, and as a result the cells are in fact cuboidal or columnar, not unlike their in vivo counterparts and conventional fluorescence microscopy on this material is generally unsatisfactory. On the other hand, with the confocal system it is often possible to reveal structures that are completely obscured in conventional fluorescence microscopy. Thus, the distinction between specific staining of different intracellular organelles, and the distinction between organelle staining versus plasma membrane staining is greatly enhanced in polarized cells. The confocal unit also has the unique ability to optically section cells either vertically or horizontally. In addition, the highly sensitive detection system of the requested microscope enables the investigator to illuminate with, and detect, low light levels. This permits us to work with living cells which normally do not survive intense illumination. The acquisition of this instrument at Georgetown will significantly enhance our ability to carry out immunofluorescence studies that are already funded by a number of NIH funded scientists in the Department of Anatomy and Cell Biology that are now sharing one fluorescence microscope. This unit is almost always occupied and as a result our research progress has been adversely affected since we do not have ready access to the microscope. Furthermore, when it breaks down our scientists are placed in a particularly stressful situation as, for most of us, there is not a suitable second microscope available for our use. The acquisition of the second fluorescence unit is essential for the successful pursuit of our research on the NIH funded projects that are listed in this grant application.
