Abstract Electrophysiology is a vital tool in neuroscience research with increasing translational value. It is used to record or modulate neuronal activity with high temporal but lower spatial resolution. Optical technologies, such as two-photon microscopy (TPM) can complement electrophysiological recordings with large-scale imaging at cellular resolution. Combining these two provides a powerful platform to elucidate and coordinate multimodal functions. Prior attempts have been limited to the superficial brain from a top-down optical view. Here, we describe a novel combination of transparent microelectrode arrays (MEAs) with glass microprisms for simultaneous electrophysiology and optical imaging of all cortical layers in a vertical plane. We tested our device in Thy1-GCaMP6 mice for over 4 months and demonstrated its capability for multisite single-unit recording, microstimulation, and simultaneous TPM calcium imaging. Using this setup, we reveal how amplitude, frequency, and depth of microstimulation impact neural activation patterns across the cortical column. This work presents a multimodal tool that extends integrated electrophysiology and optical imaging from the superficial brain to the whole cortical column, opening new avenues of neuroscience research and neurotechnology development.

Integration of microprism and microelectrode arrays enables simultaneous electrophysiology and two-photon imaging across all cortical layers