Multiphoton Intravital Microscopy to Study Lymphocyte Motility in Lymph Nodes

Intravital microscopy (IVM) allows for the direct in vivo visualization of dynamic biological processes in their physiological context at high spatial and temporal resolution. Novel nonlinear optical imaging modalities, most prominently multiphoton microscopy, have extended the spectrum of cellular functions amenable to IVM investigation to include migration and cell–cell interactions occurring deep inside the highly light-scattering environments of solid tissues, which had so far been inaccessible to conventional microscopy techniques. This has led to important new insights into immune cell behavior at steady state, as well as their change in behavior during an immune response. Here, we describe in detail a technique that allows for the monitoring of lymphocyte motility in the lymph nodes of mice at the single cell level using multiphoton intravital microscopy (MP-IVM).