Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes by two-photon microscopy

Two-photon microscopy of live tissue imaging provides insightful information about the four-dimensional dynamics of cell behavior and has contributed to the discoveries of new biological mechanisms including those in the immunology field. In recent years, it has become easier for many researchers to perform the tissue imaging experiments, due to the refinement of the commercially available microscope systems. However, it is still crucial for the efficient visualization of biological events to optimize the sample preparation by using the best available reagents. Further, it is equally important to elicit key information from the large quantity of imaging data, whose handling is often complex and laborious. Here, by taking as an example the two-photon analysis of lymphocyte migration and interaction in explanted lymph nodes, we describe the key points that need to be considered for the successful experimental design, sample preparation, image acquisition, and data analysis.