In vivo fluorescence microscopy of neuronal activity in three dimensions by use of voltage-sensitive dyes.

We report in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The measurements have approximately 16-microm spatial and 3-ms temporal resolution and reach depths of 150 microm below the cortical surface. The depth-dependent differential-fluorescence optical sections of activity are consistent with known cortical architecture and represent an important step toward in vivo measurement of functioning complex neural networks. Our observations employ a custom gradient-index lens probe and voltage-sensitive dye fluorescence; the use of epi-illumination rather than dark-field illumination provides the dramatic signal-to-noise improvement necessary for fast three-dimensional imaging.