Fluorescent and Bioluminescent Calcium Indicators with Tuneable Colors and Affinities

Abstract

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

Emerging Trends in Fluorescence Bioimaging of Divalent Metal Cations Using Small‐Molecule Indicators

“…Recently, Johnsson and coworkers reported a series of fluorogenic Ca 2 + indicators MaPCas (e. g. MaPCa-656 High , 13 in Figure 1c) with indirect fusions. [25] They connected BAPTA to the lactam-forming rhodamines through a benzene sulfonamide. The fluorogenic MaPCa-656 High shows a turn-on ratio of 120 folds after binding with HaloTag and further turned on 6 folds after Ca 2 + binding.…”

“…With the increasing sophistication of fluorophore chemistry, the Lavis lab and the Johnsson lab has recently demonstrated fluorogenic versions of targetable Ca 2 + indicators that enable no-wash Ca 2 + imaging. [23,25] These indicators preferentially exist in the non-fluorescent lactam state in cytoplasm, but shift to the open fluorescent state after binding with HaloTag). The fluorescence will be further turned on after Ca 2 + binding.…”

“…Recently, Johnsson and coworkers reported a series of fluorogenic Ca 2+ indicators MaPCas (e. g. MaPCa‐656 High , 13 in Figure 1c) with indirect fusions [25] . They connected BAPTA to the lactam‐forming rhodamines through a benzene sulfonamide.…”

“…Likewise, installing the chloroalkane HaloTag ligand on a fluorescein‐based Zn 2+ indicator ZIMIR‐HaloTag [31c] ( 26 in Figure 3) and other red [31d] ( RhoCa‐Halo , 21 in Figure 3) or far‐red [23] ( 11 in Figure 3) rhodamine‐based Ca 2+ indicators via a polyethylene glycol (PEG) linker created useful tools in diverse colors for detecting different ions in targeted subcellular structure. With the increasing sophistication of fluorophore chemistry, the Lavis lab and the Johnsson lab has recently demonstrated fluorogenic versions of targetable Ca 2+ indicators that enable no‐wash Ca 2+ imaging [23,25] …”

A long-wavelength xanthene dye for photoacoustic imaging

“…This low coupling results in little effect on the molar extinction coefficient (ε), quantum yield (Φ), or shape of the excitation/emission spectra of the dye. This 10' atomic substitution method has been used to design bright, long wavelength fluorescent dyes for various purposes like protein labeling, [12][13] sensing, [14][15] and functional imaging. [16][17][18] Xanthene dyes have been previously explored for PA applications.…”