Screening of Vibrational Spectroscopic Voltage Indicator by Stimulated Raman Scattering Microscopy

Abstract Genetically encoded voltage indicators (GEVIs) have significantly advanced voltage imaging, offering spatial details at cellular and subcellular levels not easily accessible with electrophysiology. In addition to fluorescence imaging, certain chemical bond vibrations are sensitive to membrane potential changes, presenting an alternative imaging strategy; however, challenges in signal sensitivity and membrane specificity highlight the need to develop vibrational spectroscopic GEVIs (vGEVIs) in mammalian cells. To address this need, a vGEVI screening approach is developed that employs hyperspectral stimulated Raman scattering (hSRS) imaging synchronized with an induced transmembrane voltage (ITV) stimulation, revealing unique spectroscopic signatures of sensors expressed on membranes. Specifically, by screening various rhodopsin‐based voltage sensors in live mammalian cells, a characteristic peak associated with retinal bound to the sensor is identified in one of the GEVIs, Archon, which exhibited a 70 cm ⁻¹ red shift relative to the membrane‐bound retinal. Notably, this peak is responsive to changes in membrane potential. Overall, hSRS‐ITV presents a promising platform for screening vGEVIs, paving the way for advancements in vibrational spectroscopic voltage imaging.