In vivo imaging with two-photon microscopy is becoming an indispensable technique to investigate cellular and subcellular phenomenon in living tissues including the central nervous system. This microscopy enables to image dynamics of molecules, morphology, and excitability with minimal invasion to tissues. Microglia are residual immune-responsive cells in the central nervous system and show highly dynamic response to the environmental alterations. Diverse roles of microglial functions in the intact and pathological brain are still largely unknown. In this chapter we describe the detailed method to image the dynamics of microglia in the mouse brain in vivo.