We propose to obtain an Olympus BX-50 microscope equipped with epifluorescence, phase, and DIC optics along with an imaging system capable of acquiring and analyzing high-resolution black-and-white images and lower resolution video rate color images. Specifically, we propose to obtain a MetaMorph Imaging System with a Princeton Instruments Pentamax CCD camera and a Dage MTI three-chip color CCD camera. The MetaMorph system has Windows-based software operated on a Pentium PC. The configuration of the proposed Olympus microscope has been designed to facilitate its operation in a multi-user facility and for the projects of four major users. Two CCD cameras are proposed in order to accommodate the needs of the user labs for both high-resolution, high-sensitivity image acquisition as well as video-rate color imaging. Applications range from imaging of static fixed sections with DIC or immunofluorescence, to following mitochondrial movement within living plant cells, to observing transfer of fluorescent proteins during Drosophila mating. The computer interface is designed to allow rapid image acquisition and sufficient memory to store a stack of sequential images for further manipulation. Images can be superimposed to enhance signals or to locate the position of fluorescent antibodies within cells. The microscope will be equipped with a Z motor so that images of sequential planes of focus can be acquired. The computer can also control the illumination wavelength by selecting the appropriate filter cubes. Shutters on the microscope will be controlled by the software so that image acquisition can be synchronized with their opening. In samples which may be photobleached or otherwise damaged, this feature is important to observe the same specimen during development by time-lapse. The Metamorph software package allows analysis of numerous features of objects. Among the applications of the users are measurement of cell, nuclei, and organelle number, comparison of fluorescen t intensities of different objects and areas, comparison of object shape and distribution in different cells or regions of cells, etc. Some users also wish to produce videotape movies of living cells of plants, Drosophila, yeast, and nematodes. The software package will also be used to process images derived from an existing confocal microscope, particularly enhancing its capability in 3-D reconstruction and morphometric analysis. Four major users with diverse projects plan to use the equipment. Shapes, numbers, size, interactions, fusion, splitting, volume, and membrane potential of plant organelles labelled with green fluorescent protein(GFP) and other probes will be examined in different tissues at various times in development, with particular attention to male reproductive organs. Mutants with defective pollen development will optically sectioned to determine the stage of developmental arrest. The destination of GFP-labelled accessory gland proteins will be observed in the female fruit fly following transfer from the male during mating. GFP fusions with a Drosophila nuclear envelope protein, YA, will be used to image the presence and movement in nuclei during the rapid cell cycles of living embryos. Partitioning defective mutants of C. elegans will be used to understand how the polarity of the zygote is established after fertilization and how the differences in spindle orientations in the early blastomeres are controlled. Cytoplasmic flow rates in these mutants will be analyzed by following the trajectories of cytoplasmic particles. The localization and distribution of Drosophila proteins involved in chromosome segregation during cell division will be examined in vivo. Mutants with aberration in the accurate segregation of chromosomes to daughter cells will be characterized.
