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A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies

Authors
Journal
Cell Calcium
0143-4160
Publisher
Elsevier
Publication Date
Volume
21
Issue
6
Identifiers
DOI: 10.1016/s0143-4160(97)90055-5

Abstract

Abstract We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca 2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record lines-cans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

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