LASER SCANNING CONFOCAL MICROSCOPY FACILITY

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During the last four years, a laser scanning confocal microscopy facility has been established in the Laboratory of Cellular Hematology. Confocal microscopy is a state-of-the-art technology that enhances the contrast and resolution of microscopic images by rejecting out-of-focus information. Thin optical sections of a specimen are obtained, each image is stored on a computer, and the image series is reconstructed as a stereo or 3- dimensional image. The facility is currently used for the following projects: 1. The role of neutrophil cytoskeleton and integrin molecules on adherence strength. Adhesion strength of neutrophils to glass is quantified in a parallel plate flow chamber with a tapered gap width in which a constant shear stress gradient on the strength is evaluated in the presence and absence of: a) antibodies to integrin molecules, and b) cytochalasin D, an agent which inhibits elongation of the cytoskeletal protein, F-actin. F- actin is specifically stained with fluorescent phalloidin and the adherent cell F-actin network is imaged with the confocal microscope. Precise measurements of cell thickness and F-actin distribution are made under conditions of a constant shear stress gradient. 2. Myeloid membrane antigen location during leukocyte migration. Fluorescent monoclonal antibodies to neutrophil plasma membrane adhesion molecules are used to detect the location of the molecules on neutrophils that have migrated in vitro through polycarbonate membranes t chemoattractants. Results of this study were presented at the 1992 Armed Forces Institute of Pathology Quantitative Histopathology Conference.