Multiphoton microscopy has changed how we visualize neurons by providing high-resolution, non-invasive imaging capability deep within intact brain tissue. Multiphoton imaging will likely play a major role in understanding how the brain works at the level of neural circuits. In this talk, in vivo structural and functional imaging of mouse brain using long wavelength excitation and three-photon microscopy will be presented. By quantitative comparison to two-photon microscopy, the application space where 3-photon microscopy outperforms conventional 2-photon microscopy will be defined. In addition, a number of interesting directions, including new laser sources, new spectral windows, optimum illumination schemes, etc., will be presented, and their impact on further improving the imaging depth, volume, or speed in biological tissues will be discussed.