The movie above shows the FRET channel of a microscopy experiment in which we imaged the Immunological Synapse formed between a T-cell and a glass-supported lipid bilayer serving as surrogate antigen presenting cell. As explained in the illustration on top, cell-associated TCRs are decorated with an antibody fragment conjugated with AF555 (FRET donor). Bilayer-embedded pMHCs are labeled in low abundance with AF647 (FRET acceptor). Monitoring the duration of single molecule FRET events, which appear temporarily as flashes of light and which represent single TCR-pMHC interactions, allows to determine the off-rate of synaptic binding .
time frame: 165ms per image