With the increasing availability of reliable ultra-short pulsed near infrared (700nm to 1050nm) lasers, two-photon laser scanning microscopy is now possible on a commercial basis. Considerably lower photo-toxicity can accompany the use of longer wavelength illumination, allowing long-term imaging of biological tissue. Fluorescent dyes with absorption maxima < 400nm (e.g. Fura and Indo) can be excited by near-infrared laser light. Generally the Fura dyes are preferred over the equivalent Indo versions since the latter are less fluorescent and have problems with photo-isomerisation and photo-bleaching. Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF and Furaptra were characterized using a tuneable (750nm to 850nm) 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. The Fura dyes displayed the same Ca²⁺-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 810nm, was used to determine the effective dissociation constants (Keff) and dynamic ranges (Rf) of dyes: Fura-2, -4F, -6F, -FF and Furaptra. 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 (100μM) extracellular [Ca²⁺]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10min) Ca²⁺ imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photo-damage or photo-bleaching of the dye.