Fast, cell-resolution, contiguous-wide two-photon imaging to reveal functional network architectures across multi-modal cortical areas

Fast and wide field-of-view imaging with single-cell resolution, high signal-to-noise ratio, and no optical aberrations have the potential to inspire new avenues of investigations in biology. However, such imaging is challenging because of the inevitable tradeoffs among these parameters. Here, we overcome these tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast-scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of ∼16,000 neurons at 7.5 Hz from a 9 mm2 contiguous image plane, including more than 10 sensory-motor and higher-order areas of the cerebral cortex in awake mice. Network analysis based on single-cell activities revealed that the brain exhibits small-world rather than scale-free behavior. The FASHIO-2PM is expected to enable studies on biological dynamics by simultaneously monitoring macroscopic activities and their compositional elements. [Display omitted] •High optical invariant was achieved with resonant-based two-photon microscopy•A 3 mm square of the mouse cortex can be scanned at 7.5 Hz for calcium imaging•Activity of over 16,000 neurons across multiple areas was observed•Network analysis revealed small-world properties: cost-effective cortical dynamics Ota et al. present fast and wide field-of-view two-photon microscopy with practically no optical aberrations. Combining high-performance large lenses and devices and a fast laser-scanning engine enables the recording of >16,000 neurons in awake mice. Functional network analysis with single-cell resolution reveals the small-world connectivity of the cortex.