A transparent miniscope-integrated ultrasound platform enables concurrent focused neuromodulation and wide-field imaging in freely moving mice
Hyunjoo Lee, Yehhyun Jo, Ho-Jeong Kim, Keonghwan Oh, Yongbin Ko, Jihong Lim, Hong Hanh Nguyen, Subeen Kim, Sohmyung Ha, Joo Min Park
2026 · 10.21203/rs.3.rs-10048956/v1
Abstract
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.
Abstract via europepmc.
Exposures
Exposure 1: M2 stimulation with rTAL-M2 module
Target: motor cortex — “secondary motor cortex (M2)”
Device: custom-built · SonoMod transparent LiNbO3 ultrasound transducer with refractive-index-matched transparent acoustic lens (rTAL-M2 / rTAL-RSC)
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 9,200 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | 2.5, 10, 40swept | ✓✓✓⚑ |
| Duty cycle (%) | 50 | ✓✓✓ |
| Sonication duration (s) | 0.4 | ✓✓✓⚑ |
| Free-field pressure (kPa) | 380 | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 5.4 | ✓✓✓⚑ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
400 ms pulse trains at 50% duty cycle, 9.2 MHz carrier, delivered with an inter-stimulation interval of 20 s; PRF 2.5, 10 and 40 Hz tested at M2.
Exposure 2: RSC stimulation with rTAL-RSC module (also used as off-target sham for M2)
Target: retrosplenial cortex — “retrosplenial cortex (RSC)”
Device: custom-built · SonoMod transparent LiNbO3 ultrasound transducer with refractive-index-matched transparent acoustic lens (rTAL-M2 / rTAL-RSC)
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 9,200 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | ⚑ |
| Duty cycle (%) | 50 | ✓✓✓ |
| Sonication duration (s) | 0.4 | ✓✓✓ |
| Free-field pressure (kPa) | 380 | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 5.4 | ✓✓✓ |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
400 ms pulse trains at 50% duty cycle, 9.2 MHz carrier, delivered with an inter-stimulation interval of 20 s; PRF 2.5, 10 and 40 Hz tested at M2.
Flags from extraction
n_subjects— 51 mice used in total per Methods; analyses report n=3 to 5 per comparison.exposures[0].free_field.isppa_w_cm2— Paper writes 'measured acoustic intensity was 5.4 W/cm2 (0.38 MPa)' without naming the intensity metric; Methods say hydrophone data were converted to peak pressure and ISPPA. Recorded as free-field Isppa.exposures[1].timing.pulse_repetition_frequency_hz— PRF at the RSC target is not stated separately; the 2.5/10/40 Hz sweep is described for M2.exposures[0].timing.pulse_repetition_frequency_hz— Thermal characterisation methods mention a PRF of 2.5 Hz and, elsewhere, 3 kHz; in vivo PRFs taken from the stimulation protocol paragraph.exposures[0].timing.sonication_duration_s— 400 ms is the pulse train duration; thermal tests used a 5 s train.exposures[0].device.family— Transducer designed in-house; LiNbO3 disk supplied by an external optics firm (Precision Micro-Optics), so custom_built.