A LONG-WORKING-DISTANCE MINIATURE TWO-PHOTON MICROSCOPE ENABLES CROSS-SPECIES IMAGING FROM RODENTS TO NON-HUMAN PRIMATES

Unrestrained neural imaging across species—from rodents to non-human primates—is essential for understanding the circuit mechanisms underlying complex behaviors and cognition. However, current miniature two-photon microscopes (m2PMs) are limited by short working distances (WDs) and insufficient adaptability to non-human primates, restricting their utility in neuroscience. Here, we developed a long-WD m2PM system featuring three interchangeable objectives (2 and 3 mm WD), enabling high-resolution deep brain imaging in mice and marmosets during free behavior, and in head-unrestrained macaques. In mice, integration with a small cannula enables 700 μm volumetric imaging from CA1 to the dentate gyrus (DG) with axon-level resolution and a 600×500 μm field of view (FOV), while preserving hippocampal integrity. A fast axial-focusing module further allows simultaneous imaging of CA1 and DG neurons in freely moving mice. In marmosets, an optimized cable protection design for the m2PM enables stable calcium imaging of auditory cortical neurons during naturalistic auditory behavior. In macaques, integration of the m2PM with a miniature XYZ motion stage and high-efficiency fluorescence collection enables variable-FOV, high–signal-to-noise, and robust neuronal imaging. These capabilities of long-WD m2PM enable high-resolution, deep-brain, and flexible in vivo imaging across species, providing a foundation for comparative studies of brain function at neuronal resolution across species.