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Am J Physiol Cell Physiol 279: C1278-C1284, 2000;
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Vol. 279, Issue 4, C1278-C1284, October 2000

SPECIAL COMMUNICATION
Excitation wavelengths for fura 2 provide a linear relationship between [Ca2+] and fluorescence ratio

Bradley M. Palmer1 and Russell L. Moore2

1 Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405; and 2 Department of Kinesiology and Applied Physiology, University of Colorado at Boulder, Boulder, Colorado 80309

We proposed and tested the use of nontraditional excitation wavelengths (lambda 1 and lambda 2) and an emission wavelength (lambda em) to define conditions under which free calcium concentration and a fluorescence ratio are linearly related. Fluorescence spectra were determined for aqueous solutions that contained 25 µM fura 2, 125 mM K+, and either 0 mM or 0.1 mM Ca2+. Effectively linear relationships between [Ca2+] and a fluorescence ratio, i.e., <5% bias when [Ca2+] <=  5 × dissociation constant, were apparent when lambda 1 >=  400 nm, lambda 2 <=  370 nm, and lambda em >=  510 nm. Combinations with longer lambda 1 and lambda em and/or with shorter lambda 2 reduced this bias further. Although the method described does not obviate the complications that surround the correction for fluorescence background, choosing a nontraditional combination of excitation and emission wavelengths offers several practical advantages over more traditional fura 2 fluorescence methodologies in a variety of experimental settings.

intracellular calcium; cardiac myocyte


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