Abstract
Fluorescence Recovery After Photobleaching (FRAP) is a popular and versatile family of methods used to estimate mobility and reaction parameters in cellular systems. Part of an area containing a fluorescently labeled species is bleached using a laser, and the effect of the perturbation of the spatial concentration profile of the fluorescent species is monitored. Subsequently, the collected data is reconciled with a model of the dynamics, thus yielding estimates for the parameters of interest. While originally devised to elucidate transport parameters, it was soon extended to the estimation of reaction rates, and is also used nowadays to answer a variety of questions on the organization of cellular systems.In this chapter, we review a variety of different approaches, classifying them according to the sources of uncertainty that are addressed or ignored, and the type of parameter that they attempt to estimate. We would like to highlight the importance of the general methodology as a tool that can be widely applied to a large number of situations.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, and W. W. Webb. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J, 16(9):1055–69, 1976.
J. Beaudouin, F. Mora-Bermudez, T. Klee, N. Daigle, and J. Ellenberg. Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins. Biophys J, 90(6):1878–94, 2006.
D. A. Berk, F. Yuan, M. Leunig, and R. K. Jain. Fluorescence photobleaching with spatial fourier analysis: measurement of diffusion in light-scattering media. Biophys J, 65(6):2428–36, 1993.
K. Braeckmans, L. Peeters, N. N. Sanders, S. C. De Smedt, and J. Demeester. Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope. Biophys J, 85(4):2240–52, 2003.
J. Braga, J. M. Desterro, and M. Carmo-Fonseca. Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol Biol Cell, 15(10):4749–60, 2004.
J. Braga, J. G. McNally, and M. Carmo-Fonseca. A reaction-diffusion model to study rna motion by quantitative fluorescence recovery after photobleaching. Biophys J, 92(8):2694–703, 2007.
S. R. Chary and R. K. Jain. Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc Natl Acad Sci U S A, 86(14):5385–9, 1989.
M. B. Elowitz, M. G. Surette, P. E. Wolf, J. B. Stock, and S. Leibler. Protein mobility in the cytoplasm of escherichia coli. J Bacteriol, 181(1):197–203, 1999.
M. F. Garcia-Parajo, G. M. Segers-Nolten, J. A. Veerman, J. Greve, and N. F. van Hulst. Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. Proc Natl Acad Sci U S A, 97(13):7237–42, 2000.
B. N. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien. The fluorescent toolbox for assessing protein location and function. Science, 312(5771):217–24, 2006.
N. Klonis, M. Rug, I. Harper, M. Wickham, A. Cowman, and L. Tilley. Fluorescence photobleaching analysis for the study of cellular dynamics. Eur Biophys J, 31(1):36–51, 2002.
U. Kubitscheck, P. Wedekind, and R. Peters. Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope. Biophys J, 67(3):948–56, 1994.
M. Kumar, M. S. Mommer, and V. Sourjik. Mobility of cytoplasmic, membrane, and dna-binding proteins in escherichia coli. Biophys J, 98(4):552–9, 2010.
J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy. Studying protein dynamics in living cells. Nat Rev Mol Cell Biol, 2(6):444–56, 2001.
M. S. Mommer and D. Lebiedz. Modelling subdiffusion using reaction diffusion systems. SIAM J. on Appl. Math., 70(1):112–132, 2009.
R. Peters, A. Brunger, and K. Schulten. Continuous fluorescence microphotolysis: A sensitive method for study of diffusion processes in single cells. Proc Natl Acad Sci U S A, 78(2):962–966, 1981.
R. D. Phair and T. Misteli. High mobility of proteins in the mammalian cell nucleus. Nature, 404(6778):604–9, 2000.
G. Rabut, V. Doye, and J. Ellenberg. Mapping the dynamic organization of the nuclear pore complex inside single living cells. Nat Cell Biol, 6(11):1114–21, 2004.
I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos. Effects of organelle shape on fluorescence recovery after photobleaching. Biophys J, 89(3):1482–92, 2005.
N. C. Shaner, G. H. Patterson, and M. W. Davidson. Advances in fluorescent protein technology. J Cell Sci, 120(Pt 24):4247–60, 2007.
D. Sinnecker, P. Voigt, N. Hellwig, and M. Schaefer. Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry, 44(18):7085–94, 2005.
B. A. Smith and H. M. McConnell. Determination of molecular motion in membranes using periodic pattern photobleaching. Proc Natl Acad Sci U S A, 75(6):2759–63, 1978.
D. M. Soumpasis. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J, 41(1):95–7, 1983.
B. L. Sprague and J. G. McNally. Frap analysis of binding: proper and fitting. Trends Cell Biol, 15(2):84–91, 2005.
B. L. Sprague, R. L. Pego, D. A. Stavreva, and J. G. McNally. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J, 86(6):3473–95, 2004.
T. T. Tsay and K. A. Jacobson. Spatial fourier analysis of video photobleaching measurements. principles and optimization. Biophys J, 60(2):360–8, 1991.
A. Tsuji and S. Ohnishi. Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state. Biochemistry, 25(20):6133–9, 1986.
M. Wachsmuth, T. Weidemann, G. Muller, U. W. Hoffmann-Rohrer, T. A. Knoch, W. Waldeck, and J. Langowski. Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys J, 84(5):3353–63, 2003.
M. Weiss. Challenges and artifacts in quantitative photobleaching experiments. Traffic, 5(9):662–71, 2004.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Beaudouin, J., Mommer, M.S., Bock, H.G., Eils, R. (2013). Experiment Setups and Parameter Estimation in Fluorescence Recovery After Photobleaching Experiments: A Review of Current Practice. In: Bock, H., Carraro, T., Jäger, W., Körkel, S., Rannacher, R., Schlöder, J. (eds) Model Based Parameter Estimation. Contributions in Mathematical and Computational Sciences, vol 4. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-30367-8_7
Download citation
DOI: https://doi.org/10.1007/978-3-642-30367-8_7
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-30366-1
Online ISBN: 978-3-642-30367-8
eBook Packages: Mathematics and StatisticsMathematics and Statistics (R0)