Polymer-based flexible multi-shank probes for simultaneous intracortical microstimulation and two-photon calcium imaging

Intracortical microstimulation is an essential tool for basic neuroscience and sensory restoration, yet the spatiotemporal effects of advanced multielectrode stimulation strategies on cortical networks remain poorly understood. This study presents a versatile experimental platform featuring polymer-based, flexible multi-shank electrode arrays integrated with a custom high-density neurostimulator. The polyimide penetrating probes contain densely spaced iridium oxide microelectrodes on thin, flexible shanks with sharp tips designed to minimize mechanical mismatch, tissue damage, and brain dimpling. The system was validated in transgenic GCaMP6 mice through simultaneous two-photon calcium imaging and electrical stimulation in layer 2/3 of the visual cortex. Monopolar stimulation reliably evoked robust, parameter-dependent neuronal activation, with neuronal recruitment increasing as a function of current intensity and pulse duration. Bipolar stimulation produced activation patterns distinct from monopolar stimulation, with neuronal responses that were more spatially confined when bipolar electrodes were positioned in close proximity. Furthermore, static current steering—implemented by varying the current ratio between the selected electrode pair—successfully shifted the centroid of activated neuronal populations, enabling fine spatial control over the site of activation. The developed platform provides a flexible framework for investigating local neuronal responses to complex electrical stimulation paradigms, facilitating the development of neural prostheses with higher spatial precision.