Funds are requested for an instrument package to assemble a high resolution fluorescence imaging facility which will be used predominantly to visualize DNA sequences hybridized to chromosomes, cells or tissues. To facilitate the simultaneous analysis of 8 or more probes in the same specimen an existing BioRad MRC-500 laser scanning confocal microscope will be equipped with a Spectra-Physics model 3758 dye laser pumped by a Spectra-Physics model 2045-15S/3.0 3 watt UV argon ion laser, thus permitting selection of fluorochrome excitation wavelengths continuously throughout the 400-800 nm range. To do 3-D image reconstruction and 3-D image manipulation in real time the confocal microscope will be tied to a Silicon Graphics Model W-4Dl2OGTX-B graphics workstation equipped with the Voxelview image processing package from Vital Images, Inc. To enhance the imaging of very weak fluorescence signals (and thus to facilitate the use of small DNA probes) the microscope will be equipped with a cooled CCD camera (Hamamatsu) and the existing inefficient confocal image detector replace with a cooled, low noise, photomultiplier assembly (Hamamatsu). Finally, a Near-Field Optical Scanning Device will be assembled and mounted on the confocal microscope. By circumventing the diffraction limitations of a conventional light microscope, this should permit the direct optical imaging of subcellular structures and fluorescent labeled antibodies or DNA probes with a lateral resolution of 100-200 Angstroms. The research efforts of the major user group will focus on the precise 3-dimensional localization of genes and chromosomes within interphase nuclei, the mapping of cloned human and murine DNA sequences on metaphase chromosomes, the construction of a physical linkage map for each human chromosome, the application of chromosome specific probe sets to the detection of chromosome abnormalities associate with malignant diseases, and the development of a novel method of chromosome karyotyping based on fluorescence in situ hybridization.
