Coelho-Santos Vanessa, Tieu Taryn, Shih Andy Y
Seattle Children's Research Institute, Center for Developmental Biology and Regenerative Medicine, Seattle, Washington, United States.
University of Washington, Department of Pediatrics, Seattle, Washington, United States.
Neurophotonics. 2022 Jul;9(3):031918. doi: 10.1117/1.NPh.9.3.031918. Epub 2022 Jun 3.
Two-photon microscopy is a powerful tool for imaging of the mammalian brain at cellular to subcellular resolution. However, resources that describe methods for imaging live newborn mice have remained sparse. We describe a non-invasive cranial window procedure for longitudinal imaging of neonatal mice. 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 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. The resulting chronic thinned-skull window enables imaging to a typical cortical depth of 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. This protocol enables direct visualization of the developing neurogliovascular unit in the live neonatal brain during both normal and pathological states.
双光子显微镜是一种用于以细胞到亚细胞分辨率对哺乳动物大脑进行成像的强大工具。然而，描述对新生小鼠进行活体成像方法的资源仍然稀少。我们描述了一种用于新生小鼠纵向成像的非侵入性颅窗程序。我们展示了通过对P0至P12幼鼠的颅骨进行反复刮削来构建颅窗的过程。我们使用注射器针头的边缘和手术刀刀片将骨头削薄至一定厚度。然后用氰基丙烯酸酯胶水和盖玻片加固窗口，以促进至少一周的稳定性和光学通路。头帽还包括一个轻质铝制凸缘，用于在成像过程中固定头部。由此产生的慢性薄颅骨窗口能够在不破坏颅内环境的情况下对典型的皮质深度进行成像。我们重点介绍了在发育过程中测量血管结构和血流的技术，包括使用静脉示踪剂和转基因小鼠分别标记血浆和血管细胞类型。该方案能够在正常和病理状态下直接观察新生活体大脑中发育中的神经胶质血管单元。
