Two-Photon Imaging

Abstract

This chapter will provide an overview of two-photon microscopy from elements of the theory underpinning fluorescence phenomena to functioning principles of a two-photon microscope including step-by-step practical advice on how to conduct an experiment using a two-photon microscope. In this context multi-photon excitation is also taken into consideration. By reading this chapter, you will have a synopsis of the basic principles of two-photon excitation, optical sectioning, and 3D microscopy. Furthermore, fundamentals of promising advanced methods for tissue imaging available for two-photon imaging as second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are briefly described together with classical applications on deep tissue imaging and functional brain imaging.