Label‐Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy

Abstract Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage‐sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label‐free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second‐harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole‐cell voltage‐clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label‐free read‐out. These results hold promise for the design of a non‐invasive label‐free read‐out tool for electrogenic cells.