Microspectroscopy reveals mechanisms of lymphocyte activation

The immunological synapse (IS) regulates immune responses by integrating extracellular stimuli into intracellular signalling networks, which causes leukocyte differentiation and effector functions. The dynamic spatial organisation of molecules at the IS was initially characterised by wide-field fluorescence microscopy of cell conjugates and cells interacting with planar lipid bilayers. These methods showed stable supramolecular clusters of several microns in size, which were proposed to be responsible for sustained signalling and cell-cell adhesion. The recent emergence of microspectroscopy techniques with higher spatial and temporal resolution nonetheless reveals the complex dynamics of molecular reactions that mediate IS assembly and function. This review describes microspectroscopy-based in vitro experimental approaches for imaging the molecular dynamics at the IS, as well as their contributions and open questions in the field. It also describes experimental methods to obtain quantitative parameters of dynamic biochemical reactions in living cells, and discusses about the important role of quantitative imaging and theoretical science in our understanding of molecular mechanisms underlying lymphocyte activation.