During the past two decades, two-photon microscopy has become a powerful tool in neuroscience. Unlike other imaging methods like MRI, its spatial resolution is micrometric and enables the observation of structures at the subcellular scale. In this chapter, the physical principles and the way to study phenomenon occurring in the living animal are summarized. Then, we describe the methods to observe the different components of the nervous system like neurons, glia and brain microvasculature. Finally, we discuss the advantages of two-photon microscopy in neuro-oncology and in the study of diseases.