A new method is presented that combines dual fluorescence microscopy and single-particle tracking
(SPT) to investigate the impact of spatial inhomogeneities inmembraneson lateral diffusion withinLangmuir
monolayers. In contrast to previousSPTexperiments, random walks of individual fluorescent tracer particles
and the structure of the membrane are simultaneously imaged. Monolayers of 1,2-dimyristoyl-sn-glycero-
2-phosphoethanolamine (DMPE) in the liquid condensed/liquid expanded phase are used to model the
inhomogeneous plasma membrane, while the fluorescent particles embedded in the membrane serve as
model proteins. The interactions between tracers and the domain boundary and the resulting change in
their random walks are directly observed. Since this technique permits systematic SPT studies of diffusion
in inhomogeneous monolayers, its extensions to more biologically relevant monolayers are laid out as well.