Two-photon laser scanning microscopy is an ideal tool for high-resolution fluorescence imaging in intact organs of living animals. With regard to in vivo brain research, this technique provides new opportunities to study hemodynamics in the microvascular system and morphological dynamics and calcium signaling in various glial cell types. These studies benefit from the ongoing developments for in vivo labeling, imaging, and photostimulation. Here, we review recent advances in the application of two-photon microscopy for the study of blood flow and glial cell function in the neocortex. We emphasize the dual role of two-photon imaging as a means to assess function in the normal state as well as a tool to investigate the vascular system and glia under pathological conditions, such as ischemia and microvascular disease. Further, we show how extensions of ultra-fast laser techniques lead to new models of stroke, where individual vessels may be targeted for occlusion with micrometer precision.