A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM).