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

Abstract

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.

Molecular Fluorophores for Deep-Tissue Bioimaging

“…The Miller group has developed a family of voltage sensitive indicators (RhoVR) by coupling with a molecular wire to the pendant phenyl ring of X-rhodamine dyes ( Figure 3 i). 62 − 64 These indicators operated in a membrane potential-dependent PeT process, leading to large voltage sensitivities (up to 47% ΔF/F per 100 mV). Coupling with emerging high-speed 2P microscopy, modified RhoVR can provide single-trial resolution of action potentials in neurons under 2P (λ ex = 1040 nm) illumination.…”

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells

“…Moreover, the tether has to be hydrophilic enough to stay out of the membrane but lipophilic enough to diffuse passively across the plasma membrane. 23 , 59 , 63 , 76 We chose oligoethylene glycols of different lengths to identify the best tether: HaloFlippers 2 – 5 with a linear oligoethylene glycol containing 24, 16, 8, and 0 monomer repeats ( Figure 1 D). Control 6 is as tightly tethered as 5 but as hydrophilic as 3 with an ethylene glycol 16-mer.…”

“…Since the introduction of HaloTag technology, 48 the strategy has found many applications. Examples include protein labeling with synthetic ligands 48 , 51 , 52 or fluorescent dyes to study biological processes, such as redox signaling, 53 , 54 cell dynamics, 55 − 58 and protein degradation, 59 or to detect specific ions, 60 − 65 the viscosity, 22 , 66 and the membrane potential 18 , 23 inside compartments of living organisms. They were also used as covalent long-lived tethers for protein nanomechanics.…”

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Small Molecule, Permeable, Internally Redistributing for Inner Membrane Targeting Rhodamine Voltage Reporter

“…Chemical reagents and solvents (dry) were purchased from commercial suppliers and used without further purification. Synthesis of RhoVR carboxylate (compound 1) and RhoVR-tBu were carried out as previously reported 1…”