Novel lambda FRET spectral confocal microscopy imaging method.
We report a highly specific, sensitive, and robust method for analyzing fluorescence resonance energy transfer (FRET) based on spectral laser scanning confocal microscopy imaging. The lambda FRET (lambdaFRET) 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. LambdaFRET method was validated using structurally characterized FRET standards with variable linker lengths and stoichiometries designed for this purpose. LambdaFRET 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, lambdaFRET analysis was unaffected by high fluorochrome spectral overlap and cellular autofluorescence. The lambdaFRET method demonstrated outstanding performance in intra- and intermolecular FRET analysis in both fixed and live cell imaging studies.
Megías
Diego
D
Biotechnology Programme, Confocal Microscopy and Cytometry Unit, Centro Nacional de Investigaciones Oncológicas (CNIO), C/ Melchor Fernández Almagro 3, Madrid E-28029, Spain.
Marrero
Raquel
R
Martínez Del Peso
Borja
B
García
María Angel
MA
Bravo-Cordero
José-Javier
JJ
García-Grande
Aranzazu
A
Santos
Andrés
A
Montoya
María C
MC
D001426:Bacterial Proteins / Q000032:analysis; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005720:Gamma Rays; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis; D008840:Microfilament Proteins / Q000032:analysis / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D011485:Protein Binding; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D018960:Hyaluronan Receptors; D008164:Luminescent Proteins; D008840:Microfilament Proteins; C054562:yellow fluorescent protein, Bacteria; C071058:moesin; D049452:Green Fluorescent Proteins