The Massachusetts General Hospital

1917 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
more » ... 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.
doi:10.1056/nejm191704261761710 fatcat:jtrasab2ozakxi3xlphvaqbzl4