Yang, J., Koehler, K., Davis, D. M. and Burroughs, Nigel John. (2010) An improved strip FRAP method for estimating diffusion coefficients : correcting for the degree of photobleaching. Journal of Microscopy, Vol.238 (No.3). pp. 240-253. ISSN 0022-2720

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Abstract

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)omega(2)/4 pi D for a strip of width omega. 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 mu m(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 mu m(2) s(-1) for a population of cells.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history
Divisions:	Faculty of Science > Mathematics Faculty of Science > Centre for Systems Biology
Journal or Publication Title:	Journal of Microscopy
Publisher:	Wiley-Blackwell Publishing, Inc
ISSN:	0022-2720
Date:	June 2010
Volume:	Vol.238
Number:	No.3
Number of Pages:	14
Page Range:	pp. 240-253
Identification Number:	10.1111/j.1365-2818.2009.03347.x
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Royal Society (Great Britain), Engineering and Physical Sciences Research Council (EPSRC), Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Wolfson Royal Society, Medical Research Council (Great Britain) (MRC)
Grant number:	GR/S29256/01 (EPSRC), BB/D011663/1 (BBSRC), GO500563
URI:	http://wrap.warwick.ac.uk/id/eprint/5860

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