Photo-physical properties of Ca2+-indicator dyes suitable for two-photon fluorescence-lifetime recordings

We analyzed the suitability of various Ca2+-indicator dyes for quantitative two-photon fluorescence-lifetime imaging. Although fura-2, fluo-3, BTC and calcein did not show useful Ca2+-dependent lifetime changes, calcium orange, calcium green-1, oregon green-2 and -5N, as well as magnesium green allowed to quantify the Ca2+-free and Ca2+-bound dye fractions by a double-exponential lifetime analysis. For the latter dyes, we derived calibration formalisms that correct for lifetime distortions by dye impurities and Ca2+-dependent extinction coefficients.