A new generation of Ca2+ indicators with greatly improved fluorescence properties.

TLDR

The authors synthesized a new family of highly fluorescent Ca²⁺ indicators to study cytosolic free calcium in biochemical contexts. The indicators feature an 8‑coordinate tetracarboxylate chelating site linked to stilbene chromophores, with the stilbene ethylenic bond incorporated into a heterocyclic ring to boost quantum efficiency and photostability. Compared with quin2, the dyes are up to 30‑fold brighter, exhibit wavelength shifts upon Ca²⁺ binding, have slightly lower affinity, longer excitation wavelengths, and superior Ca²⁺ selectivity, making them ideal for intracellular imaging in single cells, cell layers, and tissues.

Abstract

A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, quin2, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.