Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics

Two-photon (2P) microscopy is a high-resolution imaging technique that was initially applied by neurobiologists and developmental cell biologists but has subsequently been broadly adapted by immunologists. The value of 2P microscopy is that it affords an unparalleled view of single-cell spatiotemporal dynamics deep within intact tissues and organs. As the technology develops and new transgenic mice and fluorescent probes become available, 2P microscopy will serve as an increasingly valuable tool for assessing cell function and probing molecular mechanisms. Here we discuss the technical aspects related to 2P microscope design, explain in detail various tissue imaging preparations, and walk the reader through the often daunting process of analyzing multidimensional data sets and presenting the experimental results.