Intravital 2P-microscopy enables the longitudinal study of brain tumor biology in superficial mouse cortex layers. Intravital microscopy of the white matter, an important route of glioblastoma invasion and recurrence, has not been feasible, due to low signal-to-noise ratios and insufficient spatiotemporal resolution. Here, we present an intravital microscopy and artificial intelligence-based analysis workflow (Deep3P) that enables longitudinal deep imaging of glioblastoma up to a depth of 1.2 mm. We find that perivascular invasion is the preferred invasion route into the corpus callosum and uncover two vascular mechanisms of glioblastoma migration in the white matter. Furthermore, we observe morphological changes after white matter infiltration, a potential basis of an imaging biomarker during early glioblastoma colonization. Taken together, Deep3P allows for a non-invasive intravital investigation of brain tumor biology and its tumor microenvironment at subcortical depths explored, opening up opportunities for studying the neuroscience of brain tumors and other model systems.

活体 2P 显微镜能够对小鼠浅层皮质层的脑肿瘤生物学进行纵向研究。由于信噪比低和时空分辨率不足，白质的活体显微镜检查是胶质母细胞瘤侵袭和复发的重要途径，目前尚不可行。在这里，我们提出了一种活体显微镜和基于人工智能的分析工作流程 (Deep3P)，可以对深度达 1.2 毫米的胶质母细胞瘤进行纵向深度成像。我们发现血管周围侵袭是胼胝体的首选侵袭途径，并揭示了胶质母细胞瘤在白质中迁移的两种血管机制。此外，我们观察白质浸润后的形态变化，这是早期胶质母细胞瘤定植期间成像生物标志物的潜在基础。总而言之，Deep3P 允许对脑肿瘤生物学及其在皮层下深度的肿瘤微环境进行非侵入性活体研究，为研究脑肿瘤和其他模型系统的神经科学提供了机会。