Confocal microscopes provide superior optical sectioning, without the effect of out-of-focus light that usually compromises the quality and usefulness of light images, especially those using fluorescent probes. A series of confocal images at increasing depths in the specimen is processed for 3D reconstruction of objects labeled with fluorescent dyes or visualized by reflection, using software provided by the manufacturer plus software written in this facility. Our Leica confocal microscope incorporates an upright microscope, and has an argon-krypton laser for improved double and triple fluorescent labeling. It has two reflected/epifluorescent light detectors, and on transmitted light, non-confocal detector. Images are stored on disk during the working day, and either transferred to the users own computer or one of our image-analysis workstations using FTP, or written to optical disk for archival storage, so that the hard disk can be cleared for other users. Users are introduced to the instrument, and usually for one or two sessions, the operation is done entirely by laboratory personnel, unless of course the user already has confocal experience. Subsequently, users operate the instrument under supervision, and eventually can work alone. Sets of rules help to protect the instrument from incorrect or careless use. The instrument is housed in the resource and thus always is under the control and supervision of resource personnel. During the current grant year, we have had a total of 13 different laboratories making significant use of the confocal microscope. This represented approximately 22 different projects, and 4 institutions. Usage is very high, with the instrument in use during the day, at night, and often on weekends. We are seeking to obtain an additional instrument to relieve this heavy load. We had to replace our argon/krypton laser during the current year as the original tube failed. We also replaced the PC computer that forms the operator interface, because of intermittent software failures that we could not track down, but that disappeared when we made a temporary substitution of the entire computer. A new computer was purchased. 8-31-96
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