Novel lambda FRET spectral confocal microscopy imaging method

KEY WORDS FRET; spectral; microscopy; imaging; confocal; interaction ABSTRACT We report a highly specific, sensitive, and robust method for analyzing fluores-cence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (kFRET) algorithm comprises imaging of a FRET sample at multiple emission wavelengths rendering a FRET spectrum, which is separated into its donor and acceptor components to obtain a pixel-based calculation of FRET efficiency. The method uses a novel off-line precalibration procedure for spectral bleed-through correction based on the acquisition of reference reflection images, which simplifies the method and reduces variability. kFRET method was vali-dated using structurally characterized FRET standards with variable linker lengths and stoichio-metries designed for this purpose. kFRET performed better than other well-established methods, such as acceptor photobleaching and sensitized emission-based methods, in terms of specificity, reproducibility, and sensitivity to distance variations. Moreover, kFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The kFRET method demon