Two-Photon Intravital Microscopy Animal Preparation Protocol to Study Cellular Dynamics in Pathogenesis

Two-photon intravital microscopy (2P-IVM) is an advanced imaging platform that allows the visualization of dynamic processes at subcellular resolution in vivo. Dynamic processes like cell migration, cell proliferation, cell-cell interactions, and cell signaling have an interactive character and occur in complex environments. Hence, it is of pivotal importance to study these processes in living animals, using for example 2P-IVM. 2P-IVM can be performed on a variety of tissues, from the skin of the animal to internal organs, and a variety of methods can be utilized to perform 2P-IVM on these tissues. Here, we discuss the protocols and considerations for four of those 2P-IVM methods, namely tissue explant imaging, skin imaging, surgical exposure imaging, and multi-day window imaging. We carefully compare and explain in depth how to set up each method. Lastly, in the notes section we mention some alternative solutions for the 2P-IVM methods described. In conclusion, this protocol can be used as a guide towards deciding which 2P-IVM method to use and to enable the setup of this method.