Immune responses occur as a result of stochastic interactions between a plethora of different cell types and molecules that regulate the migration and function of innate and adaptive immune cells to drive protection from pathogen infection. The trafficking of immune cells into peripheral tissues during inflammation and then subsequent migration to draining lymphoid tissues has been quantitated using radiolabelled immune cells over 40 years ago. However, how these processes lead to efficient immune responses was unclear. Advances in physics (multi-photon), chemistry (probes) and biology (animal models) have provided immunologists with specialized tools to quantify the molecular and cellular mechanisms driving immune function in lymphoid tissues through directly visualising cellular behaviours in 3-dimensions over time. Through the temporal and spatial resolution of multi-photon confocal microscopy immunologists have developed new insights into normal immune homeostasis, host responses to pathogens, anti-tumour immune responses and processes driving development of autoimmune pathologies, by the quantification of the interactions and cellular migration involved in adaptive immune responses. Advances in deep tissue imaging, including new fluorescent proteins, increased resolution, speed of image acquisition, sensitivity, number of signals and improved data analysis techniques have provided unprecedented capacity to quantify immune responses at the single cell level. This quantitative information has facilitated development of high-fidelity mathematical and computational models of immune function. Together this approach is providing new mechanistic understanding of immune responses and new insights into how immune modulators work. Advances in biophysics have therefore revolutionised our understanding of immune function, directly impacting on the development of next generation immunotherapies and vaccines, and is providing the quantitative basis for emerging technology of simulation-guided experimentation and immunotherapeutic design.