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

• Published in: Pflügers Archiv 1995-03, Vol.429 (5), p.672-681
• Main Authors: Helm, P J, Franksson, O, Carlsson, K
• Format: Article
• Language: English
• Subjects: Calcium - metabolism; Cells, Cultured; Diffusion; Fluorescence; Fura-2; Lasers; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; Neurons - metabolism; Neurons - ultrastructure; Ultraviolet Rays
• ISSN: 0031-6768; 1432-2013
• DOI: 10.1007/BF00373988

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 mumol.l-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.