Biomaterial Database

Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching. 2005

E B Van Munster, and G J Kremers, and M J W Adjobo-Hermans, and T W J Gadella

Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands. munster@science.uva.nl

Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult due to the low excitation intensities. In addition, the method is sensitive to inadvertent donor bleaching, autofluorescence and bleed-through of excitation light. In the method introduced in this paper, donor and acceptor intensities are monitored continuously during acceptor photobleaching. Subsequently, curve fitting is used to determine the FRET efficiency. The method was demonstrated on cameleon (YC2.1), a FRET-based Ca(2+) indicator, and on a CFP-YFP fusion protein expressed in HeLa cells. FRET efficiency of cameleon in the presence of 1 mm Ca(2+) was 31 +/- 3%. In the absence of Ca(2+) a FRET efficiency of 15 +/- 2% was found. A FRET efficiency of 28% was found for the CFP-YFP fusion protein in HeLa cells. Advantages of the method are that it does not require complete acceptor photobleaching, it includes correction for spectral cross-talk, donor photobleaching and autofluorescence, and is relatively simple to use on a normal wide-field microscope.

UI	MeSH Term	Description	Entries
D008164	Luminescent Proteins	Proteins which are involved in the phenomenon of light emission in living systems. Included are the "enzymatic" and "non-enzymatic" types of system with or without the presence of oxygen or co-factors.	Bioluminescent Protein,Bioluminescent Proteins,Luminescent Protein,Photoprotein,Photoproteins,Protein, Bioluminescent,Protein, Luminescent,Proteins, Bioluminescent,Proteins, Luminescent
D008856	Microscopy, Fluorescence	Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye.	Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D006367	HeLa Cells	The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays.	Cell, HeLa,Cells, HeLa,HeLa Cell
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D031541	Fluorescence Resonance Energy Transfer	A type of FLUORESCENCE SPECTROSCOPY using two FLUORESCENT DYES with overlapping emission and absorption spectra, which is used to indicate proximity of labeled molecules. This technique is useful for studying interactions of molecules and PROTEIN FOLDING.	Forster Resonance Energy Transfer
D038761	Photobleaching	Light-induced change in a chromophore, resulting in the loss of its absorption of light of a particular wave length. The photon energy causes a conformational change in the photoreceptor proteins affecting PHOTOTRANSDUCTION. This occurs naturally in the retina (ADAPTATION, OCULAR) on long exposure to bright light. Photobleaching presents problems when occurring in PHOTODYNAMIC THERAPY, and in FLUORESCENCE MICROSCOPY. On the other hand, this phenomenon is exploited in the technique, FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING, allowing measurement of the movements of proteins and LIPIDS in the CELL MEMBRANE.