Monitoring membrane potential with second-harmonic generation

This protocol describes the nonlinear optical phenomenon known as second-harmonic generation (SHG) and discusses its special attributes for imaging membrane-potential changes in single cells and multicellular preparations. Undifferentiated N1E-115 mouse neuroblastoma cells are used as a model cellular system for membrane electrophysiology. Styryl and naphthylstyryl dyes, also known as hemicyanines, are a class of electrochromic membrane-staining probes that have been used to monitor membrane potential by fluorescence; they also produce SHG images of cell membranes with SHG intensities that are sensitive to voltage. These experiments allow for the precise characterization of the voltage sensitivity of SHG and identification of the optimal wavelength for the incident laser fundamental light. This protocol presents the steps for the culture, staining, patching, and imaging of cells. The details of the imaging system and the measurements obtained are discussed, as are the prospects of this technology for imaging membrane potential changes in neuronal preparations.