To advance our understanding of cellular processes, it would be desirable to obtain information beyond mere localisation of a labelled protein. Recent advances in microscopy have made it possible to visualise in both space and time the biochemistry of individual cells. Fluorescence lifetime imaging (FLIM) is a powerful technique for cellular imaging and is widely regarded as the most robust method of measuring Förster Resonance Energy Transfer (FRET) to study protein-protein interactions or protein conformational changes. We visualise the supramolecular organization of receptor phosphorylation using FLIM to report FRET (Treanor et al. (2006) J Cell Biol 174:1, 153-161). This broadly applicable technique can be used to image protein phosphorylation of any green fluorescent protein (GFP)-tagged receptor at an intercellular contact. Specifically, we examine the phosphorylation of Killer Ig-like Receptor (KIR) by measuring FRET between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine mAb. Visualization of KIR phosphorylation in Natural Killer (NK) cells contacting target cells expressing cognate Major Histocompatibility Complex (MHC) class I proteins revealed that KIR signalling is spatially restricted to the intercellular contact. This explains how NK cells can respond appropriately when simultaneously surveying susceptible and resistant target cells. Surprisingly, contrary to an expected homogeneous distribution of KIR signalling across the intercellular contact, phosphorylated KIR was confined to microclusters within the large aggregate of KIR. Thus, the spatial confinement of receptor phosphorylation within the intercellular contact may influence how NK cells integrate activating and inhibitory signals. In addition, developing new bio-imaging methods that can probe the microenvironment of proteins in live cells may help in our understanding of diverse aspects of cell biology such as membrane architecture, receptor trafficking, and endocytosis. Recently, we reported on the ability of FLIM to report the local refractive index of GFP in solution (Suhling et al. (2002) Biophys J 83:6, 3589-95). We further examined the application of FLIM to probe the microenvironment of GFP-tagged proteins at the cell surface and at the intercellular contact between NK cells and target cells, where a complex molecular architecture known as the immunological synapse (IS) forms. Following a novel quantitative analysis of fluorescence lifetime images, we report that the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is within the expected statistical range (Treanor et al. (2005) J Microscopy 217:1, 36-43). However, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Intriguingly, the lifetime of GFP-tagged MHC class I protein is shortened at the inhibitory NK cell IS compared to the unconjugated membrane. This likely reveals a distinct local refractive index for MHC protein clustered at the IS. This first example of the use of FLIM to probe the local environment of an IS indicates how FLIM may be broadly useful in imaging membrane heterogeneity or complexes of proteins and lipids such as those involved in IS formation. One future challenge will be to understand how to interpret refractive index changes within live cells and whether such changes can be correlated to specific membrane microdomains.