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New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.

Original publication

DOI

10.1002/bies.201100086

Type

Journal article

Journal

Bioessays

Publication Date

05/2012

Volume

34

Pages

369 - 376

Keywords

Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Microscopy, Fluorescence, Molecular Imaging, Protein Interaction Mapping