Reinforced thinned-skull window for repeated imaging of the neonatal mouse brain

Significance: Two-photon microscopy is a powerful tool for in vivo imaging of the mammalian brain at cellular to subcellular resolution. However, resources that describe methods for imaging live newborn mice have remained sparse. Aim: We describe a non-invasive cranial window procedure for longitudinal imaging of neonatal mice. Approach: We demonstrate construction of the cranial window by iterative shaving of the calvarium of P0 to P12 mouse pups. We use the edge of a syringe needle and scalpel blades to thin the bone to ∼15-μm thickness. The window is then reinforced with cyanoacrylate glue and a coverslip to promote stability and optical access for at least a week. The head cap also includes a light-weight aluminum flange for head-fixation during imaging. Results: The resulting chronic thinned-skull window enables in vivo imaging to a typical cortical depth of ∼ 200 μm without disruption of the intracranial environment. We highlight techniques to measure vascular structure and blood flow during development, including use of intravenous tracers and transgenic mice to label the blood plasma and vascular cell types, respectively. Conclusions: This protocol enables direct visualization of the developing neurogliovascular unit in the live neonatal brain during both normal and pathological states.