In Vivo Non linear Microscopy has been devised in the '90s by the Corneil group of W.W. Webb and exploit the non-linear excitaiton of fluorescence from a number of cromophores, particularly GFP mutants but also flavin proteins and many dyes.
We use pulsed (100fs) Near Infrared lasers (700-1000 nm) to prime the fluorescence. Since it is a two-photon process (i.e. non linear) the excitation is limited to the focal volume and the technique is intrisically capable of 3D optical sectioning the sample. An example of the possibility to follow cells (lymphocytes) within the lynph nodes is given in this movie. Moreover the use of infrared radiation allows to have deeper penetretation within the biological specimens. Finally the wide two-photon excitation bands allow for simultaneous excitation of more than one dye with a single laser line.