Multi‐Photon Imaging

Multi-photon microscopy, now in its twentieth year, has developed into one of the most robust and powerful techniques for live cell and in vivo fluorescence imaging. Although its theoretical framework is nearly a century old, it has only become a practical tool for biological research with the development of ultrafast lasers and scanning microscopy techniques. In this unit, we outline the basic principles of multi-photon microscopy, paying special attention to technical considerations for biological applications. Furthermore, we discuss some common applications of the technique, mainly in the field of live cell and in vivo imaging. We illustrate how multi-photon microscopy can be utilized to address questions ranging from structural cell changes to trafficking of membrane proteins in living organisms, often with resolutions of hundreds of milliseconds. We conclude by outlining the necessary elements needed to establish a successful two-photon microscopy system.