A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.
We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca2+ 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 line-scans 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.
Parker
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Department of Psychobiology, University of California Irvine 92697-4550, USA. iparker@uci.edu
Callamaras
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Wier
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WG
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000493:pharmacokinetics; D005260:Female; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009865:Oocytes / Q000737:chemistry / Q000502:physiology; D055095:Optics and Photonics / Q000295:instrumentation; D012984:Software; D014981:Xenopus
D002118:Calcium