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

Abstract

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) 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 Ca(2+) 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 Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-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 Ca(2+) probes and discuss their utility for biological Ca(2+) 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 Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) 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.

Deep two-photon brain imaging with a red-shifted fluorometric Ca 2+ indicator

“…For example, one of the early red-shifted Ca 2+ indicators, calcium orange (23), was successfully used in an in vitro Drosophila photoreceptor preparation (24), although in isolated cardiac cells its performance was quite poor compared with, for example, OGB-1 (25). In neuronal preparations, calcium orange was hardly usable in brain slices (13) or under in vivo conditions. Similar difficulties were encountered with calcium orange in zebrafish two-photon imaging recordings (26).…”

“…By using the red-shifted low-affinity (10-12). Although numerous nonfunctional fluorescent probes were developed and successfully used to image neuronal morphologies at higher cortical depths, the synthesis of an efficient long-wavelength Ca 2+ indicator dye to measure rapid neuronal activity has remained a challenge, as discussed extensively by Oheim et al (13). For example, one of the early red-shifted Ca 2+ indicators, calcium orange (23), was successfully used in an in vitro Drosophila photoreceptor preparation (24), although in isolated cardiac cells its performance was quite poor compared with, for example, OGB-1 (25).…”

“…A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths (10)(11)(12). Although optimized red-shifted genetically encoded Ca 2+ indicators are becoming increasingly available (3), red-shifted synthetic Ca 2+ indicators mostly suffer from serious limitations, including a low signal-to-noise ratio in the relevant range of Ca 2+ concentrations, slow kinetics, photobleaching, low solubility in aqueous solutions, or poor cellular dye retention (13). Here, we report that Cal-590, a red-shifted variant of the recently described fluorescent Ca 2+ indicator Cal-520 (14), greatly improved our ability to perform in vivo two-photon imaging of neuronal activity in all layers of the mouse cortex, down to layers 5 and 6 at depths of up to −900 μm below the pia.…”

An optical method to evaluate both mass and functional competence of pancreatic α- and β-cells

“…ZRL1 can also be combined with transgenic mice in which pancreatic δ-cells are labeled with EGFP. Under such a circumstance, in addition to the visualization of the three-dimensional assembly of α-, β-and δ-cells within the islets, the functions of all islet cells in the physiological-relevant environments can also be evaluated with far-red Ca 2+ fluorophores (Oheim et al, 2014).…”

Sensing Inside the Living Cells

“…Many probes respond to calcium ions by the change in intensity of a single fl uorescence band (Oheim et al 2014 ). Their response to calcium depends not only in ion binding but also on their local concentration.…”