Comprehensive quantitative evaluation of FLIM-FRET Microscopy

摘要

Average lifetime between the usually bi-exponential double-label specimen and a mono-exponential single donor sample serves as a basis for the calculation of the average energy transfer efficiency (E). This semi-quantitative approach however does not fully explore cellular functions, such as endosomal pH differences, specific morphological features, examining sub-populations and the like. We applied a different, quantitative Region-of-Interest (ROI)-based method in 2 live-cell assays by TCSPC FLIM-FRET microscopy: a 5 amino-acid linked FRET standard and mouse pituitary cells expressing a dimerized C/EBPa-bZip transcription factor in the nucleus, both tagged with Cerulean (C) and Venus (V). ROIs with different selection thresholds were generated and compared. Average lifetimes are similar, but ratios between them and other subtle differences are revealed by comprehensive distribution information. Following published references, we also explored 3 different methods to calculate FLIM-FRET energy transfer efficiencies for the Cerulean-Venus constructs, producing differences and supporting the long-held notion that E is called 'apparent' efficiency. FRET's greatest contribution continues to be exploring changes taking place at the cellular level and quantifying differences in relative terms between control and variables.