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 Ca<sup>2+</sup>
indicators like GCaMP to provide simultaneous voltage and Ca<sup>2+</sup>
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.