A Compact Two‐Photon Module for Simultaneous Dual‐Depth, Multi‐Region in Vivo Brain Imaging

ABSTRACT Understanding how distributed brain regions interact across cortical depths is crucial for uncovering the neural basis of behavior and cognition. Two‐photon microscopy (2PM) excels at imaging the superficial layer, while three‐photon microscopy (3PM) penetrates deep tissues but lacks sufficient speed for capturing fast superficial dynamics. Here, we present a compact two‐photon imaging module (2PIM) that seamlessly integrates with existing 3PM to achieve simultaneous, high‐speed imaging of superficial and deep brain regions, while preserving the deep tissue penetration of 3PM. This dual‐modality system allows for remote and independent control of imaging planes in both axial and lateral dimensions. Leveraging this technology, we discovered context‐dependent modulation of hippocampal CA1 and visual cortex responses to identical tactile stimuli, layer‐specific temporal dynamics in sensory cortex during air puff stimulation, and depth‐divergent neuronal activity patterns in motor cortex across acute‐to‐chronic pain. These results highlight the system's potential for dissecting the spatiotemporal coordination of Multi‐Regions Imaging, three‐photon microscopy, and two‐photon microscopy of distributed brain circuits.