A transparent miniscope-integrated ultrasound platform enables concurrent focused neuromodulation and wide-field imaging in freely moving mice

<title>Abstract</title> <p>Simultaneous ultrasound stimulation and optical imaging in freely behaving animals remains challenging because existing systems are often bulky, incompatible with optical readout, or used in paradigms in which stimulation and recording are temporally decoupled. Here we present an open-source transparent miniscope-integrated focused-ultrasound interface, named SonoMod, for concurrent ultrasound neuromodulation and mesoscale calcium imaging during unimpeded animal behavior. We develop an optically transparent ultrasound transducer (TUT) with a miniaturized refractive-index-matched transparent acoustic lens (rTAL) module, enabling high-resolution focused ultrasound targeting through the optical path while preserving optical access to the dorsal cortex. This module is designed to easily dock with the open-source UCLA Miniscope v4 platform, facilitating implementation in established miniscope workflows. We benchmark the electrical, acoustic, and optical performance of the system, demonstrate sensory-evoked wide-field cortical calcium activity and validate region-specific ultrasound-evoked excitatory responses at two anatomically distinct cortical targets — secondary motor cortex (M2) and retrosplenial cortex (RSC) — during naturalistic behavior. This work establishes a practical method for combining focused ultrasound stimulation with simultaneous wide-field optical readout in a package with full mobility, which will enable advances in behavioral neuroscience and therapeutic applications.</p>