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, we show that endogenous second harmonic generation (SHG) from live cells is highly sensitive to changes in membrane potential. Simultaneous electrophysiological control of a living (HEK293T) cell, through whole-cell voltage clamp reveals a linear relation between the SHG intensity and membrane voltage. Our 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. We further provide a conceptual framework that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the membrane potential 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.

Three-dimensional imaging of helicoidal pitch and structural characterization of cholesteric cellulose nanocrystals suspensions using second harmonic generation microscopy

“…In our experimental configuration, we employed a mode-locked pulsed laser (Insight DS+Spectra-Physics) that generated pulses of 80 MHz repetition rate and a 120 fs duration. 17 The excitation beam was horizontally polarized and set at a frequency of 900 nm. The generated laser output power was 1.2 Watts and had a beam diameter of 1.2 mm.…”