Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes: Monitoring Membrane Potential in the Infrared

Functional imaging microscopy based on voltage-sensitive dyes (VSDs) has proven effective for revealing spatio-temporal patterns of activity in vivo and in vitro. Microscopy based on two-photon excitation of fluorescent VSDs offers the possibility of recording sub-millisecond membrane potential changes on micron length scales in cells that lie upwards of one millimeter below the brain's surface. Here we describe progress in monitoring membrane voltage using two-photon excitation (TPE) of VSD fluorescence, and detail an application of this emerging technology in which action potentials were recorded in single trials from individual mammalian nerve terminals in situ. Prospects for, and limitations of this method are reviewed.