A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2

Abstract

A confocal scanning laser microscope (CSLM) for observation and quantitative ratiometric measurements of the intracellular dynamics of Ca2+ ions in living neurons has been developed. The instrument consists of a UV-enhanced CSLM, an optical arrangement providing simultaneous excitation at two wavelengths, an electronic arrangement for processing the simultaneous fluorescence response, and software for computing the absolute Ca2+ concentrations, ([Ca2+]). The instrument can be used for any excitation ratiometric measurements, provided that the dye substance used is excitable by wavelengths between 334 nm and 750 nm (such as, e.g. Fura-2). The spatial resolution of the CSLM, as well as a temporal resolution of 20 ms per line (maximum sampling rate) for dynamic measurements are provided by the instrument. Using Fura-2 in calibrated Ca2+ buffer solutions, the instrument measures [Ca2+] between 0 and 1.35 μmol·1−1 with an error of less than 1%. The capability of the instrument to measure absolute [Ca2+] was verified by recording fluorescence images of test solutions with well defined [Ca2+] values (Molecular Probes, Eugene, Ore., USA, C-3009 calibration solutions). In order to verify the dynamic capability of the instrument in real biological specimens, fluorescence changes of Fura-2 that were due to an intracellular flux of Ca2+ ions, and to an increase of [Ca2+]i (the intracellular Ca2+ concentration) have been recorded in Fura-2-loaded cultured cells of the line TE 671.

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Correspondence to Paul Johannes Helm.

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Helm, P.J., Franksson, O. & Carlsson, K. A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2. Pflügers Arch. 429, 672–681 (1995). https://doi.org/10.1007/BF00373988

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Key words

  • UV-enhanced confocal scanning laser microscope (UV-(SLM)
  • UV-CLSM
  • Intensitymodulated multiple-beam scanning microfluorometry (IMS)
  • Fura-2
  • Calcium
  • Calcium imaging
  • Ca2+ diffusion
  • Intracellular calcium