A functional brain vascular supply is crucial for delivery of life-essential nutrients and removal of metabolites. Abnormal vascular development and hemodynamics can result in pathologies of many vascular diseases. The enlarged high-flow blood vessels that shunt blood from arteries to veins can cause arteriovenous malformation (AVM) and it can lead to life-threatening ruptures in the brain. The ability to correlate the relationship between blood flow with vascular structure at cell levels in living animals would foster our knowledge of the disease. Conventional wide-field microscopy is powerful in imaging at cell levels; however, light penetration depth is the limitation in deep tissue imaging. Near-infrared fluorescence imaging system can achieve deep tissue imaging but the low spatial resolution makes it difficult to map vascular structure and blood flow. In this research, we use two-photon laser scanning microscopy that can achieve deep tissue imaging and high spatial resolution to do cerebral vascular imaging in the genetic mutant mice that showing the phenotype of AVM. By using the line-scan imaging and 3D scan, we are able to analyze the blood velocity and diameter of the blood vessel that give the dynamic information of blood flow and vascular structure. We hope the findings through “5D” two-photon microscopy imaging that include high spatial vascular structure (3D) and blood velocity (4th dimension) over a period of time (5th dimension) can improve insights for the mechanism of AVM formation in the brain.