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A Comment on using FLIM with FRET
Published online by Cambridge University Press: 14 March 2018
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Depending on the nature of the study and what sort of information one is trying to gather through the use of FRET, FLIM has some compelling advantages in certain situations, and can provide a quantitative evaluation of the donor, acceptor and FRET pair stoichiometry. It does require access to specialized equipment and software. Different approaches to FLIM data acquisition have different strengths and weaknesses. For dynamic studies requiring high time resolution, FLIM acquisition times can fall well short of ideal.
If a yes/no answer to whether FRET is occurring is all that is required, then the polarization anisotropy of the acceptor can be used to determine FRET between fluorescent proteins (Rizzo and Piston, 2005). This is a relatively simple and robust method for confirming the presence/absence of FRET.
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- Microscopy 101
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- Copyright © Microscopy Society of America 2006
References
Mattheyses, Hoppe, and Axelrod, (2004). 'Polarized Fluorescence Resonance Energy Transfer Microscopy.' Biophysical Journal
87:2787–2797.Google Scholar
Rizzo, and Piston, (2005). 'High-Contrast Imaging of Fluorescent Protein FRET by Fluorescence Polarization Microscopy' Biophysical Journal: Biophysical Letters L14-L16.Google ScholarPubMed
Thaler, Koushik, Blank, and Vogel, (2005). 'Quantitative Multiphoton Spectral Imaging and its use for Measuring Resonance Energy Transfer.' Biophysical Journal
89:2736–2749.CrossRefGoogle ScholarPubMed
Zimmermann, Rietdorf, Girod, Georget, and Pepperkok, (2002). 'Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.' FEBS Letters 531: 245–249.Google ScholarPubMed
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