New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging

• Choices among red-emitting Ca2+ probes have expanded with new chemical indicators and the red-emitting GECIs.
• A careful evaluation of which fluorophores work best together with EGFP is required.
• Sequential dual-cube measurements allow more fluorophore-specific detection but trade speed against spectral separability.
• Control of Ca2+ localization and local indicator concentration and Ca2+ buffer capacity will be critical.

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca2+) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca2+ probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca2+ probes. In response to this demand more red-emitting chemical and FP-based Ca2+-sensitive indicators have been developed since 2009 than in the thirty years before.In this topical review, we survey the physicochemical properties of these red-emitting Ca2+ probes and discuss their utility for biological Ca2+ imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3–16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca2+-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca2+ indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

For cell biological studies, there is an urgent need for long-wavelength Ca2+ indicators since many molecular biology and transgenic tools extensively incorporate green-emitting fluorophores and optogenetics and photochemical uncaging make use of the blue end of the spectrum. In the present review, after a summary of the prerequisites for building a Ca2+ sensor, we present the known good Ca2+ chelators and the chemistry developed to insert them into red-emitting fluorescent dyes. We give a systematic critical appraisal of the different molecules presently available and also, the pros and cons of chemical small-molecules (fast kinetics) vs. genetically encoded red-emitting Ca2+ probes (unequalled intensity and reduced photobleaching). Finally, we suggest that cargo-constructs for locally up-concentrating Ca2+ sensors in proximity of putative Ca2+ signaling sites, e.g., near the mouth of open Ca2+ channel, will improve our understanding of spatially and temporally confined Ca2+ signals (‘microdomains’).Figure optionsDownload high-quality image (55 K)Download as PowerPoint slide