Construction of Large Cranial Windows With Nanosheet and Light-Curable Resin for Long-term Two-Photon Imaging in Mice.

In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain's curved surface. The protocol enables long-term, high-resolution, and multiscale imaging-from subcellular structures to large neuronal populations-in awake mice over several months. Key features • This protocol establishes large-scale cranial windows by combining a flexible, biocompatible PEO-CYTOP nanosheet with light-curable resin. • The large-scale cranial window provides long-term optical transparency and mechanical stability, enabling chronic in vivo imaging with minimal motion artifacts. • This approach facilitates multiscale two-photon imaging-from subcellular structures to large-scale neural networks-in awake mice.