Glioblastomas (GB) are the most malignant primary brain tumors with median survival of about 17 month despite extensive treatment. Molecular heterogeneity and the tumor microenvironment contribute to the challenges in treating GBs. Glioma-associated microglia and macrophages, comprising 30-50% of non-neoplastic cells in the microenvironment, play a crucial role in facilitating tumor growth and inducing tissue necrosis. However, the intricate interplay between these immune and glioma cells remains poorly understood.This study focuses on understanding the dynamic role of microglia during glioblastoma invasion at the tumor's invading front in corpus callosum. In order to investigate glioma and microglial dynamics, we use an autochtonous model of mesenchymal GBs employing a CRISPR/Cas mediated approach in recipient Cx3Cr1Gfp knock-in mice.Previous studies using two-photon in vivo imaging were limited to superficial cortical regions, hindering investigations into deeper brain areas like the corpus callosum. Recently, three-photon microscopy has emerged as a powerful tool to image between 1100-1600µm deep into the intact brain.By implanting chronic cortical windows and taking advantage of three photon in vivo imaging, we reveal differential regulation of microglial motility based on glioma infiltration levels in the corpus callosum. Moreover, we find distinct clusters of microglial movement in dependence to the distance of tumor cells.This approach provides insights into microglia-glioma interactions at the invading front in the corpus callosum, offering a valuable tool for studying deep-seated cellular dynamics and identifying potential therapeutic targets.