Focusing new light on brain functions: multiphoton microscopy for deep and super-resolution imaging

•The principles and the technological improvements of two-photon microscopy.•The high-peak-power laser enables in vivo imaging of the hippocampal dentate gyrus.•Adaptive optical methodologies focusing on the refractive index mismatch.•PEO-CYTOP nanosheet enables in vivo deep and wide-field brain imaging.•Two-photon STED microscopy for easy and efficient in vivo nanoimaging. Multiphoton microscopy has become a powerful tool for visualizing neurobiological phenomena such as the dynamics of individual synapses and the functional activities of neurons. Owing to its near-infrared excitation laser wavelength, multiphoton microscopy achieves greater penetration depth and is less invasive than single-photon excitation. Here, we review the principles of two-photon microscopy and its technical limitations (penetration depth and spatial resolution) on brain tissue imaging. We then describe the technological improvements of two-photon microscopy that enable deeper imaging with higher spatial resolution for investigating unrevealed brain functions.