We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.
- Förster resonance energy transfer (FRET)
↵1 Permanent address: Department of Chemistry, Colorado State University, Fort Collins, CO 80525, U.S.A.
↵2 Present address: Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
Intermolecular Associations in 2D and 3D, a Biochemical Society Focused Meeting held at University of Nottingham, 19–20 June 2003
Abbreviations used: FRET, Förster resonance energy transfer; emFRET, energy migration FRET; rFLIM, anisotropy fluorescence lifetime imaging microscopy; VFP, visible fluorescent protein; RTK, receptor tyrosine kinase; CLSM, confocal laser-scanning microscope; eGFP, enhanced green fluorescent protein; YFP, yellow fluorescent protein; CHO, Chinese hamster ovary.
- © 2003 Biochemical Society