Premium
An improved strip FRAP method for estimating diffusion coefficients: correcting for the degree of photobleaching
Author(s) -
YANG J.,
KÖHLER K.,
DAVIS D. M.,
BURROUGHS N. J.
Publication year - 2010
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2009.03347.x
Subject(s) - fluorescence recovery after photobleaching , diffusion , fluorophore , photobleaching , parametrization (atmospheric modeling) , fluorescence , chemistry , effective diffusion coefficient , population , degree (music) , analytical chemistry (journal) , biological system , thermodynamics , chromatography , physics , optics , medicine , demography , radiology , sociology , biology , magnetic resonance imaging , radiative transfer , acoustics
Summary 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 τ= m 2 w 2 /4π D 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 μm 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 μm 2 s −1 for a population of cells.