TY - JOUR
T1 - A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes
AU - Kang, Minchul
AU - Day, Charles A.
AU - Drake, Kimberly
AU - Kenworthy, Anne K.
AU - DiBenedetto, Emmanuele
N1 - Funding Information:
This work was supported by National Institutes of Health grants Nos. R01 GM073846 (to A.K.K.) and 1RO1GM068953-01 (to E.D.).
PY - 2009/9/2
Y1 - 2009/9/2
N2 - 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 confocalFRAP. Finally,wedemonstrate the accuracy of these new(to our knowledge) formulae bymeasuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.
AB - 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 confocalFRAP. Finally,wedemonstrate the accuracy of these new(to our knowledge) formulae bymeasuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.
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U2 - 10.1016/j.bpj.2009.06.017
DO - 10.1016/j.bpj.2009.06.017
M3 - Article
C2 - 19720039
AN - SCOPUS:70349589255
SN - 0006-3495
VL - 97
SP - 1501
EP - 1511
JO - Biophysical journal
JF - Biophysical journal
IS - 5
ER -