The reasons why certain vaccine adjuvants and/or delivery systems are more or less effective at inducing immune responses or promoting the preferential induction of particular types of response are unknown. While vaccine antigen discovery has benefited from a systematic approach, our limited understanding of the interactions of adjuvants with cells of the immune system has hampered rational adjuvant discovery and handicapped the development of new and more effective vaccines. It is well accepted that the component parts of the immune system do not work in isolation and their interactions occur in distinct and specialised micro- and macro-anatomical locations. Consequently, significant obstacles to the systematic investigation of adjuvant effects have been the complexity of the physiological environments that adjuvants interact with and the difficulty in directly investigating these interactions dynamically in vivo. Here we describe some of the immunological and microscopical techniques that have enabled the analysis of the immune cells and their interactions, in vivo, in real time. It is only by performing such detailed and fundamental studies in vivo that we can fully understand the cellular and molecular interactions that control the immune response. These types of systematic analyses of the events involved in adjuvant action are a prerequisite if we are truly to design, build and target vaccines effectively.