Two-photon imaging has emerged as a central technology to explore the structure and function of living nervous systems. This EMBO Practical Course will provide participants with the practical experience and theoretical knowledge needed to successfully use two-photon microscopy. In the lectures students will learn the principles of optical microscopy, laser scanning, functional fluorophores, delivery techniques, and digital image processing. The laboratory practicals will demonstrate the use of a variety of molecular probes of cell function, including calcium-sensitive dyes and functionalized fluorescent proteins. A particular emphasis will be on the application of two-photon microscopy in vivo to visualize neuronal and glial dynamics in the living brain. Students will also be exposed to dendritic imaging, showing the combination of two-photon microscopy with electrophysiology. Several mouse models of neurological diseases, including Alzheimer’s disease and multiple sclerosis are available at the host institute. Hence, we will also teach the benefits and challenges of imaging diseased areas of the nervous system. Together, these lectures and practical tutorials will cover a substantial part of what is done at the forefront of two-photon imaging. This will allow students to return to their home laboratories with a comprehensive view of recent applications that they can adapt to their own research questions.