Massachusetts General Hospital
Boston Medical and Surgical Journal
Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer ͑TRET͒ plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non-FRETing populations, but have been
... ons, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex autofluorescent background decay, described using a stretched exponential function ͑StrEF͒. We develop a generalized multi-exponential fitting algorithm ͑GeMEF͒, which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein-protein interactions and protein conformational assays in a cell-based format. Presenilin 1 ͑PS1͒ is known to be important in Amyloid Precursor Protein ͑APP͒ processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.