This proposal requests a laser scanning confocal microscope (LSCM) and image processing workstation for a group of 9 majors users and 3 minor users in six departments at the University of Michigan (& one department at Ohio University). The LSCM with image analysis will also provide an important resource for the university at large. LSCM provides a major improvement over conventional light microscopy, particularly for fluorescence imaging, with 1) increased resolution, 2) increased contrast, 3) increased fluorescence sensitivity and detectability, 4) elimination of stray light and fluorescence scatter, 5) ability to achieve features 1-4 in thick sections or specimens along with optical sectioning. In fluorescent applications, the rapid movement and short excitation time of the finely focused laser beam provide for less bleaching, less toxicity from free radical formation (important in living material) and no piping (in cylindrical cells) in addition to elimination of scatter. Increased resolution and contrast, as well as optical sectioning is also possible from Golgi stained material. Image processing is necessary to achieve any image from LSCM. Construction of 3-D images from optical sectioning and interactive tilting, rotating, slicing, and image enhancement is possible with an appropriately powerful computer workstation. LSCM with 3-D image processing provides an improved ability to analyze the morphological appearance, location and distribution of the label. These features are critical to the needs of the user group and their research programs.
