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

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Abstract
We proposed and tested the use of nontraditional excitation wavelengths (λ1and λ2) and an emission wavelength (λem) 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., 2+] ≤ 5 × dissociation constant, were apparent when λ1≥ 400 nm, λ2≤ 370 nm, and λem≥ 510 nm. Combinations with longer λ1and λemand/or with shorter λ2reduced 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.