Two‐Photon Excitation Microscopy for the Study of Living Cells and Tissues

Abstract Two‐photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three‐dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two‐photon excitation and discuss the advantages and limitations of its use in laser‐scanning microscopy. The principal advantages of two‐photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two‐photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two‐photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. Curr. Protoc. Cell Biol . 59:4.11.1‐4.11.24. © 2013 by John Wiley & Sons, Inc.