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Applications in Confocal Microscopy

Fluorescent Protein Photobleaching Literature Sources

The field of cell biology is rapidly being transformed by the application of fluorescent proteins as fusion tags to track dynamic behavior in living cells. In this regard, fluorescence recovery after photobleaching (FRAP) is often employed to selectively destroy fluorescent molecules within a region of interest with a high-intensity laser, followed by monitoring the recovery of new fluorescent molecules into the bleached area over a period of time with low-intensity laser light. The resulting information can be used to determine kinetic properties, including the diffusion coefficient, mobile fraction, and transport rate of the fluorescently labeled molecules. The selected references below point to important literature sources for information on FRAP with fluorescent proteins.

Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W., Mobility measurement by analysis of fluorescence photobleaching recovery kinetics., Biophysical Journal 16: 1055-1069 (1976). | PubMed |

Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E. L., and Podleski, T. R., Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers., Proceedings of the National Academy of Sciences USA 73: 4594-4598 (1976). | PubMed |

Beaudouin, J., Gerlich, D., Daigle, N., Eils, R., and Ellenberg, J., Nuclear envelope breakdown proceeds by microtubule induced tearing of the lamina., Cell 108: 83-96 (2002). | PubMed |

Belgareh, N., Rabut, G., Bai, S. W., van Overbeek, M., Beaudouin, J., Daigle, N., Zatsepina, O. V., Pasteau, F., Labas, V., Fromont-Racine, M., Ellenberg, J., and Doye, V., An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells., Journal of Cell Biology 154: 1147-1160 (2001). | PubMed |

Boisvert, F. M., Kruhlak, M. J., Box, A. K., Hendzel, M. J., and Bazett-Jones, D. P., The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body., Journal of Cell Biology 152: 1099-1106 (2001). | PubMed |

Braeckmans, K., Peeters, L., Sanders, N. N., De Smedt, S. C., and Demeester, J., Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope., Biophysical Journal 85: 2240-2252 (2003). | PubMed |

Braga, J., Desterro, J. M. P., and Carmo-Fonseca, M., Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes., Molecular Biology of the Cell 15: 4749-4760 (2004). | PubMed |

Brown, E. B., Shear, J. B., Adams, S. R., Tsien, R. Y., and Webb, W. W., Photolysis of caged calcium in femtoliter volumes using two-photon excitation., Biophysical Journal 76: 489-499 (1999). | PubMed |

Calapez, A., Pereira, H. M., Calado, A., Braga, J., Rino, J., Carvalho, C., Tavanez, J. P., Wahle, E., Rosa, A. C., and Carmo-Fonseca, M., The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive., Journal of Cell Biology 159: 795-805 (2002). | PubMed |

Carrero, G., McDonald, D., Crawford, E., de Vries, Gerda, and Hendzel, M. J., Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins., Methods 29: 14-28 (2003). | PubMed |

Chen, D. and Huang, S., Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells., Journal of Cell Biology 153: 169-176 (2001). | PubMed |

Cheutin, T., McNairn, A. J., Jenuwein, T., Gilbert, D. M., Singh, P. B., and Misteli, T., Maintenance of stable heterochromatin domains by dynamic HP1 binding., Science 299: 721-725 (2003). | PubMed |

Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M., and Lippincott-Schwartz, J., Diffusional mobility of golgi proteins in membranes of living cells., Science 273: 797-801 (1996). | PubMed |

Coscoy, S., Waharte, F., Gautreau, A., Martin, M., Louvard, D., Mangeat, P., Arpin, M., and Amblard, F., Molecular analysis of microscopic ezrin dynamics by two-photon FRAP., Proceedings of the National Academy of Sciences USA 99: 12813 (2002). | PubMed |

Daigle, N., Beaudouin, J., Hartnell, L., Imreh, G., Hallberg, E., Lippincott-Schwartz, J., and Ellenberg, J., Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells., Journal of Cell Biology 154: 71-84 (2001). | PubMed |

Dayel, M. J., Hom, E. F. Y., and Verkman, A. S., Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum., Biophysical Journal 76: 2843-2851 (1999). | PubMed |

Dundr, M., Hoffmann-Rohrer, U., Hu, Q., Grummt, I., Rothblum, L. I., Phair, R. D., and Misteli, T., A kinetic framework for a mammalian RNA polymerase in vivo., Science 298: 1623-1626 (2002). | PubMed |

Edidin, M., Zagyanky, Y., and Lardner, T. J., Measurement of membrane protein lateral diffusion in single cells., Science 191: 466-468 (1976). | PubMed |

Ellenberg, J., Siggia, E. D., Moreira, J. E., Smith, C. L., Presley, J. F., Worman, H. J., and Lippincott-Schwartz, J., Nuclear membrane dynamics and reassembly in living cells: Targeting of an inner nuclear membrane in interphase and mitosis., Journal of Cell Biology 138: 1193-1206 (1997). | PubMed |

Essers, J., Houtsmuller, A. B., van Veelen, L., Paulusma, D., Nigg, A. L., Pastink, A., Vermeulen, W., Hoeijmakers, J. H., and Kanaar, R., Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage., The EMBO Journal 21: 2030-2037 (2002). | PubMed |

Gerlich, D., Beaudouin, J., Gebhard, M., Ellenberg, J., and Eils, R., Four-dimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells., Nature Cell Biology 3: 852-855 (2001). | PubMed |

Gerlich, D., Beaudouin, J., Kalbfuss, B., Daigle, N., Eils, R., and Ellenberg, J., Global chromosome positions are transmitted through mitosis in mammalian cells., Cell 112: 751-764 (2003). | PubMed |

Gordon, G. W., Chazotte, B., Wang, X. F., and Herman, B., Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components., Biophysical Journal 68: 766-778 (1995). | PubMed |

Handwerger, K. E., Murphy, C., and Gall, J. G., Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle., Journal of Cell Biology 160: 495-504 (2003). | PubMed |

Haraguchi, T., Live cell imaging: Approaches for studying protein dynamics in living cells., Cell Structure and Function 27: 333-334 (2002). | PubMed |

Hirschberg, K., Miller, C. M., Ellenberg, J., Presley, J. F., Siggia, E. D., Phair, R. D., and Lippincott-Schwartz, J., Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells., Journal of Cell Biology 143: 1485-1503 (1998). | PubMed |

Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten, D., Hoeijmakers, J. H., and Vermeulen, W., Action of DNA repair endonuclease ERCC1/XPF in living cells., Science 284: 958-961 (1999). | PubMed |

Houtsmuller, A. B. and Vermeulen, W., Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching., Histochemistry and Cell Biology 115: 13-21 (2001). | PubMed |

Jacobson, K., Hou, Y., and Wojcieszyn, J., Evidence for lack of damage during photobleaching measurements of the lateral mobility of cell surface components., Experimental Cell Research 116: 179-189 (1978). | PubMed |

Klonis, N., Rug, M., Harper, I., Wickman, M., Cowman, A., and Tilley, L., Fluorescence photobleaching analysis for the study of cellular dynamics., European Biophysics Journal 31: 36-51 (2002). | PubMed |

Kohler, R. H., Cao, J., Zipfel, W. R., Webb, W. W., and Hanson, M. R., Exchange of protein molecules through connections between higher plant plastids., Science 276: 2039-2042 (1997). | PubMed |

Köster, M., Frahm, T., and Hauser, H., Nucleocytoplasmic shuttling revealed by FRAP and FLIP technologies., Current Opinion in Biotechnology 16: 28-34 (2005). | PubMed |

Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E., Bazett-Jones, D. P., and Hendzel, M. J., Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments., Journal of Cell Biology 150: 41-51 (2000).
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Kruse, C., Jaedicke, A., Beaudouin, J., Bohl, F., Ferring, D., Guttler, T., Ellenberg, J., and Jansen, R. P., Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p., Journal of Cell Biology 159: 971-982 (2002). | PubMed |

Kubitscheck, U., Tschödrich-Rsotter, M., Wedekind, P., and Peters, R., Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements., Journal of Microscopy 182: 225-233 (1996). | PubMed |

Levin, M. H., Haggie, P. M., Vetrivel, L., and Verkman, A. S., Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus., Journal of Biological Chemistry 276: 21331-21336 (2001). | PubMed |

Lippincott-Schwartz, J., The secretory membrane system studied in real-time., Histochemistry and Cell Biology 116: 97-107 (2001). | PubMed |

Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G. H., Photobleaching and photoactivation: Following protein dynamics in living cells., Nature Cell Biology Supplement: S7-S14 (2003). | PubMed |

Lippincott-Schwartz, J., Roberts, T. H., and Hirschberg, K., Secretory protein trafficking and organelle dynamics in living cells., Annual Review of Cell and Developmental Biology 16: 557-589 (2000). | PubMed |

Lippincott-Schwartz, J., Snapp, E., and Kenworthy, A., Studying protein dynamics in living cells., Nature Reviews Molecular Cell Biology 2: 444-456 (2001). | PubMed |

McDonnell, J. M., Surface plasmon resonance: Towards an understanding of the mechanisms of biological molecular recognition., Current Opinions in Chemical Biology 5: 572-577 (2001). | PubMed |

McNally, J. G. and Smith, C. L., Photobleaching by confocal microscopy., in Confocal and Two-Photon Microscopy, Diaspro, A. (ed.), Wiley-Liss, New York, pages 525-538 (2001).
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Meyvis, T. K. L., De Smedt, S. C., Van Oostveldt, P., and Demeester, J., Fluorescence recovery after photobleaching: A versatile tool for mobility and interaction measurements in pharmaceutical research., Pharmaceutical Research 16: 1153-1162 (1999). | PubMed |

Misteli, T., Protein dynamics: Implications for nuclear architecture and gene expression., Science 291: 843-847 (2001). | PubMed |

Nehls, S., Snapp, E. L., Cole, N. B., Zaal, K. J. M., Kenworthy, A. K., Roberts, T. H., Ellenberg, J., Presley, J. F., Siggia, E., and Lippincott-Schwartz, J., Dynamics and retention of misfolded proteins in native endoplasmic reticulum membranes., Nature Cell Biology 2: 288-295 (2000). | PubMed |

Nikonov, A. V., Snapp, E., Lippincott-Schwartz, J., and Kreibich, G., Active translocation complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum., Journal of Cell Biology 158: 497-506 (2002). | PubMed |

Ölveczky, B. P. and Verkman, A. S., Monte Carlo analysis of obstructed diffusion in three dimensions: Application to molecular diffusion in organelles., Biophysical Journal 74: 2722-2730 (1998). | PubMed |

Partikian, A., Ölveczky, B. P., Swaminathan, R., Li, Y., and Verkman, A. S., Rapid diffusion of green fluorescent protein in the mitochondrial matrix., Journal of Cell Biology 140: 821-829 (1998). | PubMed |

Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., and Pison, D. W., Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy., Biophysical Journal 73: 2782-2790 (1997). | PubMed |

Patterson, G. H. and Lippincott-Schwartz, J., A photoactivatable GFP for selective photolabeling of proteins and cells., Science 297: 1873-1877 (2002). | PubMed |

Pederson, T., Protein mobility within the nucleus-What are the right moves?., Cell 104: 635-638 (2001). | PubMed |

Periasamy, N., Bicknese, S., and Verkman, A. S., Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: Singlet and triplet state quenching by tryptophan., Photochemistry and Photobiology 63: 265-271 (1996).
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Periasamy, N. and Verkman, A. S., Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: Application to photobleaching measurements of multicomponent and anomalous diffusion., Biophysical Journal 75: 557-567 (1998).
| PubMed |

Phair, R. D. and Misteli, T., High mobility of proteins in the mammalian cell nucleus., Nature 404: 604-609 (2000). | PubMed |

Phair, R. D. and Misteli, T., Kinetic modelling approaches to in vivo imaging., Nature Reviews Molecular Cell Biology 2: 898-907 (2001). | PubMed |

Phair, R. D., Scaffidi, P., Elbi, C., Vecerová, J., Dey, A., Ozato, K., Brown, D. T., Hager, G., Bustin, M., and Misteli, T., Global nature of dynamic protein-chromatin interactions in vivo: Three-dimensional genome scanning and dynamic interaction networks of chromatin proteins., Molecular and Cellular Biology 24: 6393-6402 (2004). | PubMed |

Piston, D. W., Patterson, G. H., and Knobel, S. M., Quantitative imaging of the green fluorescent protein (GFP)., Methods in Cell Biology 58: 31-48 (1999). | PubMed |

Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. M., and Lippincott-Schwartz, J., Endoplasmic reticulum-to-Golgi transport visualized in living cells., Nature 389: 81-85 (1997). | PubMed |

Reits, E. A. J. and Neefjes, J. J., From fixed to FRAP: Measuring protein mobility and activity in living cells., Nature Cell Biology 3: E145-E147 (2001). | PubMed |

Sciaky, N., Presley, J., Smith, C., Zaal, K. J. M., Cole, N., Moreira, J. E., Terasaki, M., Siggia, E., and Lippincott-Schwartz, J., Golgi tubule traffic and the effects of Brefeldin A visualized in living cells., Journal of Cell Biology 139: 1137-1155 (1997). | PubMed |

Schwille, P., Kummer, S., Heikal, A. A., Moerner, W. E., and Webb, W. W., Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins., Proceedings of the National Academy of Sciences USA 97: 151-156 (2000). | PubMed |

Seiffert, S. and Oppermann, W., Systematic evaluation of FRAP experiments performed in a confocal laser scanning microscope., Journal of Microscopy 220: 20-30 (2005).
| PubMed |

Seksek, O., Biwersi, J., and Verkman, A. S., Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus., Journal of Cell Biology 138: 131-142 (1997).
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Shav-Tal, Y., Singer, R. H., and Darzacq, X., Imaging gene expression in single living cells., Nature Reviews Molecular Cell Biology 5: 855-861 (2004). | PubMed |

Siggia, E. D., Lippincott-Schwartz, J., and Bekiranov, S., Diffusion in inhomogeneous media: Theory and simulations applied to whole cell photobleach recovery., Biophysical Journal 79: 1761-1770 (2000). | PubMed |

Slepchenko, B. M., Schaff, J. C., Carson, J. H., and Loew, L. M., Computational cell biology: Spatiotemporal simulation of cellular events., Annual Review of Biophysics and Biomolecular Structure 31: 423-441 (2002). | PubMed |

Slepchenko, B. M., Schaff, J. C., Macara, I., and Loew, L. M., Quantitative cell biology with the virtual cell., Trends in Cell Biology 13: 570-576 (2003). | PubMed |

Smith, A. E., Slepchenko, B. M., Schaff, J. C., Loew, L. M., and Macara, I. G., Systems analysis of Ran transport., Science 295: 488-491 (2002). | PubMed |

Song, L., Varma, C.A., Verhoeven, J. W., and Tanke, H. J., Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy., Biophysical Journal 70: 2959-2968 (1996). | PubMed |

Sprague, B. L. and McNally, J. G., FRAP analysis of binding: Proper and fitting., Trends in Cell Biology 15: 84-91 (2005). | PubMed |

Stout, A. L. and Axelrod, D., Spontaneous recovery of fluorescence by photobleached surface-adsorbed proteins., Photochemistry and Photobiology 62: 239-244 (1995).
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Swaminathan, R., Hoang, C. P., and Verkman, A. S., Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: Cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion., Biophysical Journal 72: 1900-1907 (1997). | PubMed |

Tour, O., Meijer, R. M., Zacharias, D. A., Adams, S. R., and Tsien, R. Y., Genetically targeted chromophore-assisted light inactivation., Nature Biotechnology 21: 1505-1508 (2003).
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Verkman, A. S., Solute and macromolecule diffusion in cellular aqueous compartments., Trends in Biochemical Sciences 27: 27-33 (2002). | PubMed |

Vigers, G. P., Coue, M., and McIntosh, J. R., Fluorescent microtubules break up under illumination., Journal of Cell Biology 107: 1011-1024 (1988). | PubMed |

Wachsmuth, M., Weidemann, T., Müller, G., Hoffmann-Rohrer, U. W., Knoch, T. A., Waldeck, W., and Langowski, J., Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching., Biophysical Journal 84: 3353-3363 (2003). | PubMed |

Wedekind, P., Kubitscheck, U., and Peters, R., Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging., Journal of Microscopy 176: 23-33 (1994). | PubMed |

Wedekind, P., Kubitscheck, U., Heinrich, O., and Peters, R., Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion., Biophysical Journal 71: 1621-1623 (1996). | PubMed |

Weiss, M. and Nilsson, T., In a mirror dimly: Tracing the movements of molecules in living cells., Trends in Cell Biology 14: 267-273 (2004). | PubMed |

Wolf, D. E., Edidin, M., and Dragsten, P. R., Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method., Proceedings of the National Academy of Sciences USA 77: 2043 (1980). | PNAS |

Zaal, K. J. M., Smith, C. L., Polishchuk, R. S., Altan, N., Cole, N. B., Ellenberg, J., Hirschberg, K., Presley, J. F., Roberts, T. H., Siggia, E., Phair, R. D., and Lippincott-Schwartz, J., Golgi membranes are absorbed into and reemerge from the ER during mitosis., Cell 99: 589-601 (1999). | PubMed |