Intensity range based quantitative FRET data analysis to localize protein molecules in live cell nuclei.
Förster (fluorescence) resonance energy transfer (FRET) is an ideal technique to estimate the distance between interacting protein molecules in live specimens using intensity-based microscopy. The spectral overlap of donor and acceptor- essential for FRET-also generates a contamination of the FRET signal. There are a number of algorithms available to remove this spectral bleedthrough (SBT) contamination and in this paper we compare two popular algorithms to estimate the SBT element and to calculate a more precise level of energy transfer efficiency, and with that a more accurate distance estimate.
Chen
Ye
Y
W.M. Keck Center for Cellular Imaging, University of Virginia, Gilmer Hall, Charlottesville, Virginia 22904, USA.
Periasamy
Ammasi
A
D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D016764:Cell Polarity; D003627:Data Interpretation, Statistical; D004285:Dogs; D018274:Electroporation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010902:Pituitary Gland / Q000166:cytology; D010957:Plasmids; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism; D014162:Transfection
D022763:CCAAT-Enhancer-Binding Protein-alpha; D005456:Fluorescent Dyes; C045281:IgA receptor; D007074:Immunoglobulin G; D008024:Ligands; D008164:Luminescent Proteins; D011506:Proteins; D011961:Receptors, Fc; D049452:Green Fluorescent Proteins