Transparent microelectrode arrays integrated with microprisms for electrophysiology and simultaneous two-photon imaging across cortical layers Qianru Yang 1,2 , Bingchen Wu 1,2 , Elisa Castagnola 1 , May Yoon Pwint 1,2 , Alberto L. Vazquez 2,3,4 , X. Tracy Cui 1,2,4 1 Department of Bioengineering, University of Pittsburgh, United States. 2 Center for Neural Basis of Cognition, University of Pittsburgh and Carnegie Mellon University, United States. 3 Department of Radiology, University of Pittsburgh, United States. 4 McGowan Institute for Regenerative Medicine, University of Pittsburgh, United States. Abstract Electrophysiology is a vital tool in neuroscience research and translational treatment. It records or modulates neuronal activities with high temporal but low spatial resolutions. Optical technology such as two-photon microscopy (TPM), could complement electrophysiology with large-scale imaging at cellular resolution. Combining these two could provide a powerful platform that coordinates multimodal functions. However, 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 electrophysiology and simultaneous 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 TPM calcium imaging. Using this setup, we revealed the neuronal network activity across multiple cortical layers in response to microstimulation with varying depths and parameters. This work presents a multi-modal tool that extends integrated electrophysiology and optical imaging from the superficial brain to the whole cortical column, which will greatly advance neuroscience research and neurotechnology development. Keywords Microprism, electrophysiology, calcium imaging, microstimulation, two-photon microscopy (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted August 16, 2022. ; https://doi.org/10.1101/2022.07.08.499369 doi: bioRxiv preprint