High accuracy 4D cell tracking into explanted skin using two-photon excitation microscopy

Two-photon excitation microscopy (2PEM) analysis of large explanted organs is still laborious, principally because of tissue movements inducing lateral and axial drifts during extended imaging sessions. Here, we describe a two-step approach to track motile T cells in murine dorsal explanted skin with the best accuracy. First, we compared various explanted skin mounting methods for 2PEM analysis to define the setup allowing for minimal sample drift over time. Second, we developed two algorithms with the ImageJ software (National Institute of Health, Bethesda, MD) to correct the residual drift using lateral and axial registration of the collagen network. Finally, we applied the macro we developed to track fluorescent T cells in explanted skin. We found that our newly developed macro is more efficient than freely or commercially available software for shift correction, leading to more accurate velocity calculations. Our work provides a practical guide for investigators interested to employ skin-imaging approaches and offers a free alternative to commercial software for correcting lateral and axial drifts.