A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

被引:69
作者
Kang, Minchul [1 ]
Day, Charles A. [1 ]
Drake, Kimberly [1 ]
Kenworthy, Anne K. [1 ,2 ]
DiBenedetto, Emmanuele [1 ,3 ]
机构
[1] Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, Nashville, TN 37212 USA
[2] Vanderbilt Univ, Sch Med, Dept Cell & Dev Biol, Nashville, TN 37212 USA
[3] Vanderbilt Univ, Sch Med, Dept Math, Nashville, TN 37212 USA
基金
美国国家卫生研究院;
关键词
CORRELATION SPECTROSCOPY; LIVING CELLS; PROTEIN; DIFFUSION; DYNAMICS; MOBILITY; VISCOSITY; FRAP;
D O I
10.1016/j.bpj.2009.06.017
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.
引用
收藏
页码:1501 / 1511
页数:11
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