Fura2

Fura-2 is a fluorescent calcium indicator used to measure intracellular calcium ion concentration ([Ca2+]). It exists as a membrane-permeant acetoxymethyl ester, Fura-2 AM, which enters cells and is hydrolyzed by intracellular esterases to the active form that binds Ca2+.

Fura-2 is a ratiometric indicator. Upon binding Ca2+, its excitation spectrum shifts such that fluorescence upon excitation at 340 nm increases while excitation at 380 nm decreases; emission is around 510 nm. The ratio F340/F380 correlates with [Ca2+], allowing semi-quantitative measurement that is less dependent on dye concentration, path length, or illumination intensity.

The indicator has a dissociation constant (Kd) around 145 nM, with a useful dynamic range for physiological Ca2+ concentrations. It can report rapid Ca2+ transients in living cells when used with fluorescence microscopy or plate-based readers, enabling relative or semi-quantitative analyses of intracellular Ca2+ dynamics.

Loading and calibration: typical loading involves 2–5 μM Fura-2 AM; after hydrolysis, imaging is performed. Calibration to convert fluorescence ratios to [Ca2+] requires measurements of Rmin and Rmax using Ca2+-free and Ca2+-saturated conditions, often with Ca2+ ionophores, to define the limits of the dye’s response and apply the Grynkiewicz equation.

Limitations include the need for UV excitation and specialized optics, potential phototoxicity and dye leakage, and calibration complexity. Fura-2 has been foundational in neuroscience, cardiology, and other fields for studying cellular Ca2+ signaling, and remains widely used alongside newer indicators and derivatives.

History: Fura-2 was introduced in 1985 by Grynkiewicz, Poenie, and Tsien, as part of a family of ratiometric Ca2+ indicators that also includes Indo-1.