HIGH-YIELD TWO-PHOTON CALCIUM IMAGING IN FREELY MOVING MICE

Decoding how large-scale neuronal networks encode behavior, memory, and spatial navigation requires chronic, cellular-resolution recordings across wide brain regions in naturalistic conditions. Silicon probes provide millisecond temporal resolution but lack genetic specificity, optical sectioning, and cell-type selectivity. One-photon miniscopes allow imaging in freely moving animals but compromise optical sectioning, restricting experiments to sparsely labeled samples. Current two-photon (2P) head-mounted systems—including MINI2P (Zong et al., 2022) and the fiber-based 2P-FENDO (Accanto et al., 2023)—either image small fields of view (FOVs) or trade spatial resolution for throughput, limiting recordings to roughly hundreds of neurons. As a result, simultaneous high-resolution imaging of thousands of neurons remains beyond the capability of existing miniscopes.
Here, we present a next-generation 2P miniscope designed to approach the ten-thousand-neuron recording regime in freely moving mice. Key innovations include: (i) a high–numerical aperture (NA 0.45), large-aperture mini-objective supporting up to a 1-mm-diameter FOV—four times larger than MINI2P—while maintaining cellular resolution; (ii) a large-angle MEMS scanner, doubling the scan range of prior designs; (iii) a compact, high-bandwidth silicon photomultiplier with wide-angle collection optics; and (iv) a multi-beam spatiotemporal multiplexing module enabling simultaneous multi-plane imaging.
Using a single excitation fiber, we recorded high signal-to-noise calcium signals from ~1,000 neurons within a 1-mm FOV in layer II visual cortex of freely behaving GCaMP6s mice. This yield exceeds that of existing 2P miniscopes under comparable conditions. Spatiotemporal multiplexing with multiple excitation fibers further enables simultaneous imaging across depth-offset planes, increasing cell yield without compromising speed or signal quality.