This application requests funds to purchase a Zeiss LSM 510 Meta confocal microscope system. This new instrument will greatly extend the current capabilities for fluorescent microscopy at Princeton University. The Zeiss LSM 510 Meta system will allow researchers to image cells simultaneously labeled with multiple fluorescent probes, including fluorescent dyes and proteins whose emission spectra substantially overlap. Existing microscopes at Princeton University use optical filters and dichroic mirrors to select specific emitted wavelengths and therefore are limited to cleanly distinguishing only 2 different fluorescent proteins in the same cell (e.g. YFP and CFP, or GFP and RFP). Furthermore, when colocalizing fluorescent proteins are imaged on moving particles, spatial offsets occur because the different wavelengths are captured in temporally separate scans/exposures. Both problems lead to limitations in the scope and rate of research progress. The Zeiss LSM 510 Meta detector system solves these problems by using a diffraction grating to spread the emitted light over an array of light detectors, effectively determining the emission spectrum for every pixel in the specimen. Using previously measured reference spectra, the contribution of emitted light from each fluorophore is computed for every point in the image, allowing the spatial distribution of each fluorophore to be imaged separately. Preliminary results with biological systems currently under study at Princeton University, and published results from elsewhere, demonstrate that the Zeiss LSM 510 Meta detector will allow researchers to simultaneously image as many as eight different fluorescent proteins and dyes. This new capability will allow Princeton researchers to identify multiple cell compartments in living cells and follow the transport of rapidly moving intracellular particles. Research currently undertaken at Princeton which will be benefited greatly by a Meta system include studies of mRNA transport and cell movement in Drosophila oocytes and embryos, herpes virus assembly and transport in neuronal cells, morphogenesis of normal and cancerous ducts in breast tissue, C. elegans morphogenesis, and the cell biology of conjugation in yeast. Study of each of these biological systems will provide valuable information pertaining to significant human health problems such as birth defects, viral infection and cancer. ? ? ?
