Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca2+-free and Ca2+-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca 2+-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca2+-free and Ca2+-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (Keff) and dynamic ranges (Rf) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (Keff = 181 ± 52 nM, 1.16 ± 0.016 μM, 5.18 ± 0.3 μM, 19.2 ± 1 μM, and 58.5 ± 2 μM; and Rf = 22.4 ± 3.8, 12.2 ± 0.34, 6.3 ± 0.17, 16.1 ± 2.8, and 25.4 ± 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca2+] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 μM) extracellular [Ca2+]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca2+ imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.
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