FRETLoading... alone detects molecular proximity at 1–10 nanometers–precisely the distance scale of protein-protein interactions. But traditional intensity-based FRET measurements are confounded by expression variation: high signal could mean high FRET efficiency OR high protein levels.
FLIMLoading... solves this by measuring fluorescence lifetime–an intrinsic property that decreases when FRET occurs. Critically, this lifetime change is independent of how much protein is present. Whether a sample has abundant protein or sparse protein at an immune synapse, the lifetime measurement remains quantitative.
This combination enables detection of biologically decisive interactions that expression-based assays miss–including checkpointLoading... engagement in patients labeled "PD-L1 negative" by conventional testing.