Optical Recording of Action Potentials with Second-Harmonic Generation Microscopy

Abstract

Nonlinear microscopy has proven to be essential for neuroscience investigations of thick tissue preparations. However, the optical recording of fast (∼1 msec) cellular electrical activity has never until now been successfully combined with this imaging modality. Through the use of second-harmonic generation microscopy of primaryAplysianeurons in culture labeled with 4-[4-(dihexylamino)phenyl][ethynyl]-1-(4-sulfobutyl)pyridinium (inner salt), we optically recorded action potentials with 0.833 msec temporal and 0.6 μm spatial resolution on soma and neurite membranes. Second-harmonic generation response as a function of change in membrane potential was found to be linear with a signal change of ∼6%/100 mV. The signal-to-noise ratio was ∼1 for single-trace action potential recordings but was readily increased to ∼6–7 with temporal averaging of ∼50 scans. Photodamage was determined to be negligible by observing action potential characteristics, cellular resting potential, and gross cellular morphology during and after laser illumination. High-resolution (micrometer scale) optical recording of membrane potential activity by previous techniques has been limited to imaging depths an order of magnitude less than nonlinear methods. Because second-harmonic generation is capable of imaging up to ∼400 μm deep into intact tissue with submicron resolution and little out-of-focus photodamage or bleaching, its ability to record fast electrical activity should prove valuable to future electrophysiology studies.