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 (approximately 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 primary Aplysia neurons 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 microm 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 approximately 6%/100 mV. The signal-to-noise ratio was approximately 1 for single-trace action potential recordings but was readily increased to approximately 6-7 with temporal averaging of approximately 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 approximately 400 microm 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.