. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CONFOCAL MICROSCOPY 117. Figure 1. Dual-channel contocal imaging shows the distribution and morphological relationships of micro- glia (green) and astrocytes (red) in a double-labeled rat brain tissue slice. Microglia and blood vessels were stained with a fluorescein (FITO-conjugated isolectin. IB4 (Sigma, St. Louis: see Dailey and Waite. 1999). Astrocytes were immunohistochemically labeled with monoclonal antibodies against the glial fibrillary acidic protein (GFAP; Sigma, St. Louis. MO), followed by Cy5 conjugated secondar
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CONFOCAL MICROSCOPY 117. Figure 1. Dual-channel contocal imaging shows the distribution and morphological relationships of micro- glia (green) and astrocytes (red) in a double-labeled rat brain tissue slice. Microglia and blood vessels were stained with a fluorescein (FITO-conjugated isolectin. IB4 (Sigma, St. Louis: see Dailey and Waite. 1999). Astrocytes were immunohistochemically labeled with monoclonal antibodies against the glial fibrillary acidic protein (GFAP; Sigma, St. Louis. MO), followed by Cy5 conjugated secondary antibodies (Amersham Phar- macia). Note the astrocyte processes (end-feet) that line the surface of the blood vessels (arrows). in three dimensions. One advantage of volume rendering from confocal image stacks is that the sample can be viewed from any angle. This can help clarify the spatial relation- ships of cells and tissue structures. As an example, we show, in Figure 2, a 3-D rendering of four microglial cells adjacent to a branching blood vessel within a volume of brain tissue reconstructed from a stack of confocal optical sections. It would be very difficult to distinguish the small cellular processes adjacent to the brightly staining blood vessels with conventional widefield fluorescence microscopy. In most cases, the images generated by the confocal microscope are in digital form. Thus, a wide range of digital image processing capabilities can be applied to the data set. Features of cell and tissue structure can be quantified and analyzed with the aid of software packages that either stand alone or are already integrated into the confocal system software. Imaging the Dynamics of Cell and Tissue Development and Morphology In addition to the spatial information about cell structure, we can collect, with time-lapse confocal microscopy, infor- mation about the dynamics of cellular structure over time— from seconds to days. In many organisms, cell growth and locomotion
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