Fluorescent and bioluminescent calcium indicators with tuneable colors and affinities

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 (ER). 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. Graphical abstract