Brain is changing every moment i.e. the most dynamic organ, thus postmortem image or freeze-frame can provide limited information. Investigation of the brain needs a few levels of hierarchical analysis from molecular to cellular to system. Advances in imaging technique enables us to observe the dynamic changes in the brain. Using two-photon or other intravital optical imaging techniques have introduced to meet this demands. Here we introduce optical imaging technique for cellular and systemic levels of the brain also the cerebral hemodynamics. Regard with the cerebral hemodynamics, we can record the RBC velocity along capillary and vessel diameter changes using two-photon laser scanning microscopy through cranial window. Intrinsic optical signal imaging can provide additional change of regional cerebral blood volume. We can observe the neuronal activities and also astrocyte activation using two-photon microscopy. For systemic recording of brain, we use voltage-sensitive dye imaging. We apply these techniques to study the neuronal and hemodynamic changes in various brain diseases, such as Alzheimer’s disease and stroke. In vivo dynamic imaging technique is promising to overcome the current limitation of brain research.