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

Cell Calcium. 1997 Jun;21(6):441-52. doi: 10.1016/s0143-4160(97)90055-5.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Calcium / analysis
  • Calcium / pharmacokinetics
  • Female
  • Image Processing, Computer-Assisted / instrumentation
  • Image Processing, Computer-Assisted / methods
  • Microscopy, Confocal / instrumentation*
  • Microscopy, Confocal / methods*
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Oocytes / chemistry
  • Oocytes / physiology
  • Optics and Photonics / instrumentation
  • Software
  • Xenopus

Substances

  • Calcium