Compound metalens-based miniature two-photon microscope for large-FOV imaging in freely behaving animals
Zeyu Hao, Yao Zhang, Yufei Zhu, Bianhua Sun, Hongchen Jiao, Yanhui Hu, Zhili Lin, Lishuang Feng, Aimin Wang, Shumin Xiao, Runlong Wu
Abstract
Abstract Miniaturized two-photon microscopes (m2PMs) enable high-resolution, deep-brain imaging in freely behaving animals. However, the trade-offs among weight, form factor, and optical performance in conventional refractive objectives present major obstacles to achieving large field-of-view (FOV), high-resolution imaging in weight-sensitive or multiregion applications. Metalenses offer ultralight, high-NA, aberration-corrected alternatives, but face persistent challenges, including restricted FOV, off-axis aberrations, and dual-band optimization for two-photon imaging. Here, we present Meta-m2PM 2.0, a 1.06 g headpiece featuring a compound metalens architecture with a two-stage imaging design that decouples scanning and focusing. We designed and fabricated a large-angle (± 12°) metalens-based scan lens and a high-NA (0.48) dual-layer metalens-based objective, both derived from our established polarization-insensitive Si₃N₄ meta-atom library co-optimized for focusing and transmission at 920 nm excitation and 500–560 nm fluorescence collection. Meta-m2PM 2.0 achieves a 350 × 330 µm 2 FOV (> 50 × that of Meta-m2PM 1.0), with 1.17 µm lateral and 19.85 µm axial resolution at the center, while maintaining > 80% lateral resolution uniformity across the FOV, and extending imaging depth to 210 µm (~ 3 × deeper). We demonstrate dendritic imaging in head-fixed awake mice and, to our knowledge, the first metalens-based two-photon calcium imaging in freely behaving mice with a high signal-to-noise ratio, paving the way for ultralight multiregion neuroimaging and compact nonlinear microendoscopy.