Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio.

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 microM fura 2, 125 mM K(+), and either 0 mM or 0.1 mM Ca(2+). Effectively linear relationships between [Ca(2+)] and a fluorescence ratio, i.e., <5% bias when [Ca(2+)] </= 5 x 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.