| An improved strip FRAP method for estimating diffusion coefficients: correcting for the degree of photobleaching. | |
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MedLine Citation:
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PMID: 20579262 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Fluorescence recovery after photobleaching is a widely established method for the estimation of diffusion coefficients, strip bleaching with an associated recovery curve analysis being one of the simplest techniques. However, its implementation requires near 100% bleaching in the region of interest with negligible fluorescence loss outside, both constraints being hard to achieve concomitantly for fast diffusing molecules. We demonstrate that when these requirements are not met there is an error in the estimation of the diffusion coefficient D, either an under- or overestimation depending on which assumption is violated the most. We propose a simple modification to the recovery curve analysis incorporating the concept of the relative bleached mass m giving a revised recovery time parametrization tau=m(2)w(2)/4piD for a strip of width w. This modified model removes the requirement of 100% bleaching in the region of interest and allows for limited diffusion of the fluorophore during bleaching. We validate our method by estimating the (volume) diffusion coefficient of FITC-labelled IgG in 60% glycerol solution, D= 4.09 +/- 0.21 microm(2) s(-1), and the (surface) diffusion coefficient of a green-fluorescent protein-tagged class I MHC protein expressed at the surface of a human B cell line, D= 0.32 +/- 0.03 microm(2) s(-1) for a population of cells. |
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Authors:
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J Yang; K Köhler; D M Davis; N J Burroughs |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Journal of microscopy Volume: 238 ISSN: 1365-2818 ISO Abbreviation: J Microsc Publication Date: 2010 Jun |
Date Detail:
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Created Date: 2010-06-28 Completed Date: 2010-09-23 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0204522 Medline TA: J Microsc Country: England |
Other Details:
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Languages: eng Pagination: 240-53 Citation Subset: IM |
Affiliation:
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Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Cell Line Cell Membrane / chemistry* Diffusion* Green Fluorescent Proteins / analysis, genetics HLA-C Antigens / analysis*, genetics Humans Microscopy, Fluorescence / methods* Models, Theoretical Photobleaching Recombinant Fusion Proteins / analysis, genetics |
| Grant Support | |
ID/Acronym/Agency:
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BB/D011663/1//Biotechnology and Biological Sciences Research Council; GO500563//Medical Research Council; GR/S29256/01//Biotechnology and Biological Sciences Research Council |
| Chemical | |
Reg. No./Substance:
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0/HLA-C Antigens; 0/HLA-Cw6; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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