Pre-resonance stimulated Raman scattering spectroscopy and imaging of membrane potential using near-infrared rhodopsins

Voltage imaging has become an emerging technique to record membrane potential change in living cells. Yet, compared to the conventional electrophysiology, imaging approaches are still limited to relative membrane potential changes, losing important information conveyed by absolute value of membrane voltage. This challenge comes from several factors affecting the signal intensity, such as concentration, illumination intensity, and photobleaching. Spectroscopy is a quantitative method that shows potential to report the state of molecules in situ. Here, we apply electronic pre-resonance stimulated Raman scattering (SRS) imaging to detect near-infrared absorbing microbial rhodopsin voltage sensors in E. coli. The use of newly developed near-infrared microbial rhodopsins (Ganapathy et. al. 2017. JACS, 2017, 139(6):2338- 44) enables electronic pre-resonance SRS imaging with single cell sensitivity. By spectral profile analysis, we identified voltage-sensitive SRS peaks. The spectral signature can be used as part of a quantitative approach to measure membrane potential and enable mapping of absolute voltage in a neural network.