Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue

Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemical synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically-encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer (PeT) triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethyleneglycol (PEG) linker (RhoVR-Halos). When applied to cells, RhoVR-Halos selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halos maintain high voltage sensitivities—up to 34% ΔF/F per 100 mV—and fast response times typical of untargeted RhoVRs, while gaining the selectivity typical of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca²⁺ indicators like GCaMP to provide simultaneous voltage and Ca²⁺ imaging. In brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read out of action potentials from labeled cortical neurons in rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically-encodable systems, permitting imaging modalities inaccessible to either technique individually.