Abstract

The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.

© 2007 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. J. McNally and C. Smith, "Photobleaching by confocal microscopy," in Confocal and Two-Photon Microscopy: Foundation, Applications and Advances, A. Diaspro, ed. (Wiley-Liss, 2001), pp. 525-538.
  2. J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
    [CrossRef] [PubMed]
  3. J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
    [CrossRef] [PubMed]
  4. 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, 1055-1069 (1976).
    [CrossRef] [PubMed]
  5. T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
    [CrossRef] [PubMed]
  6. G. Carrero, D. McDonald, E. Crawford, G. de Vries, and M. J. Hendzel, "Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins," Methods 29, 14-28 (2003).
    [CrossRef] [PubMed]
  7. G. H. Patterson and J. Lippincott-Schwartz, "A photoactivatable GFP for selective photolabeling of proteins and cells," Science 297, 1873-1877 (2002).
    [CrossRef] [PubMed]
  8. M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
    [CrossRef] [PubMed]
  9. U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
    [CrossRef]
  10. I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
    [CrossRef] [PubMed]
  11. I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
    [CrossRef]
  12. D. M. Soumpasis, "Theoretical analysis of fluorescence photobleaching recovery experiments," Biophys. J. 41, 95-97 (1983).
    [CrossRef] [PubMed]
  13. E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
    [CrossRef] [PubMed]
  14. E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
    [CrossRef] [PubMed]
  15. 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, 3473-3495 (2004).
    [CrossRef] [PubMed]
  16. B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
    [CrossRef] [PubMed]
  17. M. Weiss, "Challenges and artifacts in quantitative photobleaching experiments," Traffic 5, 662-671 (2004).
    [CrossRef] [PubMed]
  18. A. Boivin and E. Wolf, "Electromagnetic field in the neighborhood of the focus of a coherent beam," Phys. Rev. 138, B1561-B1565 (1965).
    [CrossRef]
  19. G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
    [CrossRef] [PubMed]
  20. A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
    [CrossRef] [PubMed]
  21. K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
    [CrossRef] [PubMed]
  22. A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
    [CrossRef]
  23. L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
    [CrossRef] [PubMed]
  24. K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
    [CrossRef] [PubMed]
  25. J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
    [CrossRef]
  26. J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
    [CrossRef] [PubMed]
  27. A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
    [CrossRef]
  28. J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
    [CrossRef] [PubMed]
  29. C. J. R. Sheppard and P. Török, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997).
    [CrossRef]
  30. C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
    [CrossRef]
  31. C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
    [CrossRef] [PubMed]
  32. M. Velez and D. Axelrod, "Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes," Biophys. J. 53, 575-591 (1988).
    [CrossRef] [PubMed]
  33. A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
    [CrossRef] [PubMed]
  34. K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
    [CrossRef] [PubMed]
  35. U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
    [CrossRef] [PubMed]
  36. S. Gupta, B. Bhawna, P. Goswami, A. Agarwal, and A. Pradhan, "Experimental and theoretical investigation of fluorescence photobleaching and recovery in human breast tissue and tissue phantoms," Appl. Opt. 43, 1044-1052 (2004).
    [CrossRef] [PubMed]

2007 (1)

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

2006 (5)

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

2005 (4)

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
[CrossRef]

A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
[CrossRef] [PubMed]

2004 (7)

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
[CrossRef] [PubMed]

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

S. Gupta, B. Bhawna, P. Goswami, A. Agarwal, and A. Pradhan, "Experimental and theoretical investigation of fluorescence photobleaching and recovery in human breast tissue and tissue phantoms," Appl. Opt. 43, 1044-1052 (2004).
[CrossRef] [PubMed]

M. Weiss, "Challenges and artifacts in quantitative photobleaching experiments," Traffic 5, 662-671 (2004).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

2003 (2)

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

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

2002 (1)

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

2001 (1)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

2000 (1)

E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
[CrossRef] [PubMed]

1999 (3)

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

1998 (1)

U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
[CrossRef]

1997 (1)

C. J. R. Sheppard and P. Török, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997).
[CrossRef]

1995 (1)

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

1994 (1)

U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
[CrossRef] [PubMed]

1992 (1)

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

1988 (1)

M. Velez and D. Axelrod, "Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes," Biophys. J. 53, 575-591 (1988).
[CrossRef] [PubMed]

1983 (1)

D. M. Soumpasis, "Theoretical analysis of fluorescence photobleaching recovery experiments," Biophys. J. 41, 95-97 (1983).
[CrossRef] [PubMed]

1976 (1)

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, 1055-1069 (1976).
[CrossRef] [PubMed]

1965 (1)

A. Boivin and E. Wolf, "Electromagnetic field in the neighborhood of the focus of a coherent beam," Phys. Rev. 138, B1561-B1565 (1965).
[CrossRef]

Aalst, H. V.

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

Agarwal, A.

Axelrod, D.

M. Velez and D. Axelrod, "Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes," Biophys. J. 53, 575-591 (1988).
[CrossRef] [PubMed]

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, 1055-1069 (1976).
[CrossRef] [PubMed]

Barozzi, S.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

Beaudouin, J.

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

Bekiranov, S.

E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
[CrossRef] [PubMed]

Berland, K. M.

A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
[CrossRef] [PubMed]

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Bhawna, B.

Birmingham, J.

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

Blonk, J.

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

Boivin, A.

A. Boivin and E. Wolf, "Electromagnetic field in the neighborhood of the focus of a coherent beam," Phys. Rev. 138, B1561-B1565 (1965).
[CrossRef]

Braeckmans, K.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

Braga, J.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
[CrossRef] [PubMed]

Brakenhoff, G. J.

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

Brown, E. B.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

Bungay, P. M.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

Carmo-Fonseca, M.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
[CrossRef] [PubMed]

Carrero, G.

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

Chen, W.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Chirico, G.

A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
[CrossRef]

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Cianci, G. C.

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Collini, M.

A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
[CrossRef]

Corosu, M.

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

Crawford, E.

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

Daigle, N.

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

de Vries, G.

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

Demeester, J.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

Desterro, J. M. P.

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
[CrossRef] [PubMed]

Diaspro, A.

A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
[CrossRef]

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Dobrucki, J.

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Don, A.

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

Dong, C.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Dong, C. Y.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

Ellenberg, J.

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

Elson, E.

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, 1055-1069 (1976).
[CrossRef] [PubMed]

Faretta, M.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

Goswami, P.

Gupta, S.

Hayer, A.

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

Helenius, A.

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

Hendzel, M. J.

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

Hennink, E. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

Hofstraat, J. W.

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

Hsiao, C.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Jan, G.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Jee, S.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Kaplan, P. D.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

Kenworthy, A.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

Klee, T.

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

Koppel, D. E.

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, 1055-1069 (1976).
[CrossRef] [PubMed]

Koumoutsakos, P.

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

Kubitscheck, U.

U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
[CrossRef]

U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
[CrossRef] [PubMed]

Lin, S.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

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

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
[CrossRef] [PubMed]

Lo, W.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Lucas, B.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

McDonald, D.

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

McNally, J.

J. McNally and C. Smith, "Photobleaching by confocal microscopy," in Confocal and Two-Photon Microscopy: Foundation, Applications and Advances, A. Diaspro, ed. (Wiley-Liss, 2001), pp. 525-538.

McNally, J. G.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

Meyvis, T. K.

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

Mezzacasa, A.

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

Mora-Bermdez, F.

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

Mueller, F.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

Nagy, A.

A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
[CrossRef] [PubMed]

Oostveldt, P. V.

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

Patterson, G. H.

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

Peeters, L.

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

Pego, R. L.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

Peters, R.

U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
[CrossRef]

U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
[CrossRef] [PubMed]

Pradhan, A.

Ramoino, P.

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Remaut, K.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

Robello, M.

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

Rooij, G. J. V.

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

Sanders, N. N.

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

Sbalzarini, I. F.

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

Schlessinger, J.

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, 1055-1069 (1976).
[CrossRef] [PubMed]

Schneider, M.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard and P. Török, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997).
[CrossRef]

Siggia, E. D.

E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
[CrossRef] [PubMed]

Smedt, S. C. D.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

Smith, C.

J. McNally and C. Smith, "Photobleaching by confocal microscopy," in Confocal and Two-Photon Microscopy: Foundation, Applications and Advances, A. Diaspro, ed. (Wiley-Liss, 2001), pp. 525-538.

Snapp, E.

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

So, P. T. C.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

Song, L.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

Soumpasis, D. M.

D. M. Soumpasis, "Theoretical analysis of fluorescence photobleaching recovery experiments," Biophys. J. 41, 95-97 (1983).
[CrossRef] [PubMed]

Sprague, B. L.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

Stavreva, D. A.

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

Stubbe, B. G.

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

Su, J.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Sun, Y.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Tanke, H. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

Testa, I.

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

Török, P.

C. J. R. Sheppard and P. Török, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997).
[CrossRef]

Tung, C.

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

Usai, C.

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Vandenbroucke, R. E.

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

Velez, M.

M. Velez and D. Axelrod, "Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes," Biophys. J. 53, 575-591 (1988).
[CrossRef] [PubMed]

Webb, W. W.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

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, 1055-1069 (1976).
[CrossRef] [PubMed]

Wedekind, P.

U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
[CrossRef]

U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
[CrossRef] [PubMed]

Weiss, M.

M. Weiss, "Challenges and artifacts in quantitative photobleaching experiments," Traffic 5, 662-671 (2004).
[CrossRef] [PubMed]

Wolf, E.

A. Boivin and E. Wolf, "Electromagnetic field in the neighborhood of the focus of a coherent beam," Phys. Rev. 138, B1561-B1565 (1965).
[CrossRef]

Wu, E. S.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

Wu, J.

A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
[CrossRef] [PubMed]

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Young, I. T.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

Yu, B.

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

Zipfel, W.

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

Zwier, J. M.

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (16)

J. Beaudouin, F. Mora-Bermdez, T. Klee, N. Daigle, and J. Ellenberg, "Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins," Biophys. J. 90, 1878-1894 (2006).
[CrossRef] [PubMed]

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, 1055-1069 (1976).
[CrossRef] [PubMed]

M. Schneider, S. Barozzi, I. Testa, M. Faretta, and A. Diaspro, "Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region,"Biophys. J. 89, 1346-1352 (2005).
[CrossRef] [PubMed]

I. F. Sbalzarini, A. Mezzacasa, A. Helenius, and P. Koumoutsakos, "Effects of organelle shape on fluorescence recovery after photobleaching," Biophys. J. 89, 1482-1492 (2005).
[CrossRef] [PubMed]

I. F. Sbalzarini, A. Hayer, A. Helenius, and P. Koumoutsakos, "Simulations of (an)isotropic diffusion on curved biological surfaces," Biophys. J. 90, 878-885 (2006).
[CrossRef]

D. M. Soumpasis, "Theoretical analysis of fluorescence photobleaching recovery experiments," Biophys. J. 41, 95-97 (1983).
[CrossRef] [PubMed]

E. B. Brown, E. S. Wu, W. Zipfel, and W. W. Webb, "Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery," Biophys. J. 77, 2837-2849 (1999).
[CrossRef] [PubMed]

E. D. Siggia, J. Lippincott-Schwartz, and S. Bekiranov, "Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery," Biophys. J. 79, 1761-1770 (2000).
[CrossRef] [PubMed]

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, 3473-3495 (2004).
[CrossRef] [PubMed]

B. L. Sprague, F. Mueller, R. L. Pego, P. M. Bungay, D. A. Stavreva, and J. G. McNally, "Analysis of binding at a single spatially localized cluster of binding sites by fluorescence recovery after photobleaching," Biophys. J. 91, 1169-1191 (2006).
[CrossRef] [PubMed]

A. Nagy, J. Wu, and K. M. Berland, "Observation volumes and gamma-factors in two-photon fluorescence fluctuation spectroscopy," Biophys. J. 89, 2077-2090 (2005).
[CrossRef] [PubMed]

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, "Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy," Biophys. J. 68, 2588-2600 (1995).
[CrossRef] [PubMed]

K. Braeckmans, L. Peeters, N. N. Sanders, S. C. D. Smedt, and J. Demeester, "Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope," Biophys. J. 85, 2240-2252 (2003).
[CrossRef] [PubMed]

M. Velez and D. Axelrod, "Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes," Biophys. J. 53, 575-591 (1988).
[CrossRef] [PubMed]

K. Braeckmans, K. Remaut, R. E. Vandenbroucke, B. Lucas, S. C. D. Smedt, and J. Demeester, "Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples," Biophys. J. 92, 2172-2183 (2007).
[CrossRef] [PubMed]

U. Kubitscheck, P. Wedekind, and R. Peters, "Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope," Biophys. J. 67, 948-956 (1994).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

K. Braeckmans, B. G. Stubbe, K. Remaut, J. Demeester, and S. C. D. Smedt, "Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation-a case study for fluorescein," J. Biomed. Opt. 11, 044013 (2006).
[CrossRef] [PubMed]

J. Microsc. (4)

J. Blonk, A. Don, H. V. Aalst, and J. Birmingham, "Fluorescence photobleaching recovery in the confocal scanning light microscope," J. Microsc. 169, 363-374 (1992).
[CrossRef]

J. M. Zwier, G. J. V. Rooij, J. W. Hofstraat, and G. J. Brakenhoff, "Image calibration in fluorescence microscopy," J. Microsc. 216, 15-24 (2004).
[CrossRef] [PubMed]

C. J. R. Sheppard and P. Török, "Effects of specimen refractive index on confocal imaging," J. Microsc. 185, 366-374 (1997).
[CrossRef]

U. Kubitscheck, P. Wedekind, and R. Peters, "Three-dimensional diffusion measurements by scanning microphotolysis," J. Microsc. 192, 128-138 (1998).
[CrossRef]

Methods (1)

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

Microsc. Res. Tech. (4)

C. Y. Dong, B. Yu, P. D. Kaplan, and P. T. C. So, "Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin," Microsc. Res. Tech. 63, 81-86 (2004).
[CrossRef]

C. Hsiao, Y. Sun, W. Chen, C. Tung, W. Lo, J. Su, S. Lin, S. Jee, G. Jan, and C. Dong, "Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin," Microsc. Res. Tech. 69, 992-997 (2006).
[CrossRef] [PubMed]

A. Diaspro, M. Corosu, P. Ramoino, and M. Robello, "Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture," Microsc. Res. Tech. 47, 196-205 (1999).
[CrossRef] [PubMed]

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc. Res. Tech. 64, 135-141 (2004).
[CrossRef] [PubMed]

Mol. Biol. Cell. (1)

J. Braga, J. M. P. Desterro, and M. Carmo-Fonseca, "Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes," Mol. Biol. Cell. 15, 4749-4760 (2004).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (1)

J. Lippincott-Schwartz, E. Snapp, and A. Kenworthy, "Studying protein dynamics in living cells," Nat. Rev. Mol. Cell Biol. 2, 444-456 (2001).
[CrossRef] [PubMed]

Pharm. Res. (1)

T. K. Meyvis, S. C. D. Smedt, P. V. Oostveldt, and J. Demeester, "Fluorescence recovery after photobleaching: a versatile tool for mobility and interaction measurements in pharmaceutical research," Pharm. Res. 16, 1153-1162 (1999).
[CrossRef] [PubMed]

Phys. Rev. (1)

A. Boivin and E. Wolf, "Electromagnetic field in the neighborhood of the focus of a coherent beam," Phys. Rev. 138, B1561-B1565 (1965).
[CrossRef]

Q. Rev. Biophys. (1)

A. Diaspro, G. Chirico, and M. Collini, "Two-photon fluorescence excitation and related techniques in biological microscopy," Q. Rev. Biophys. 38, 97-166 (2005).
[CrossRef]

Science (1)

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

Traffic (1)

M. Weiss, "Challenges and artifacts in quantitative photobleaching experiments," Traffic 5, 662-671 (2004).
[CrossRef] [PubMed]

Other (2)

J. McNally and C. Smith, "Photobleaching by confocal microscopy," in Confocal and Two-Photon Microscopy: Foundation, Applications and Advances, A. Diaspro, ed. (Wiley-Liss, 2001), pp. 525-538.

A. Diaspro, G. Chirico, C. Usai, P. Ramoino, and J. Dobrucki, "Photobleaching," in Handbook of Biological Confocal Microscopy, 3rd ed., J. Pawley, ed. (Plenum, 2006), pp. 690-699.
[CrossRef]

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (8)

Fig. 1
Fig. 1

Homogeneity properties of the immobile fluorescent sample constituted by ionic cross-linking of fluorescently labeled polyelectrolyte chains. As shown in (a) homogeneity was verified at different locations on the sample by computing the pixel-by-pixel ratio between an object 3D stack [exemplary slices in (c)] and a reference 3D stack (b). x, y, and z scales are in micrometers. (d) Shows exemplary slices of the ratio and the respective intensity histogram: distribution is centered at 1.04 with a FWHM of 0.16. (e) Shows the approach used to analyze the homogeneity along the optical axis achieved by acquiring a 12 μm depth 3D stack and by computing the pixel-by-pixel ratio between each optical section and a reference one taken in the middle of the stack. The resulting 3D data is shown in (f) together with exemplary histograms for the first ( z = + 6 μm , blue line), the last ( z = 6 μm , red line) and an intermediate slice ( z = + 0.2 μm , green line). x, y, and z scales are in micrometers.

Fig. 2
Fig. 2

(a) Orthogonal projections of the bleach pattern obtained by bleaching a circular region with a radius of 0 .1 μm in confocal microscopy. The bleach pattern was produced by delivering 1 .2 mW on the sample with scanning speed v = 0.384 μm / s and distance between adjacent lines Δ y = 0.0075 μm . x, y, and z scales are in micrometers. The bleach scan was iterated ten times. (b) Orthogonal projections of the bleach pattern obtained by bleaching a circular region with a radius of 0 .1 μm in two-photon microscopy. The bleach pattern was produced by delivering an average power of 3 .8 mW with scanning speed v = 0.384 μm / s and distance between adjacent lines Δ y = 0.0075 μm . x, y, and z scales are in micrometers. The bleach scan was iterated 12 times. (c) Fluorescent sample immobility test: by bleaching a square region of 0 .8 μm a mobile fraction of less than 10% is observed, which recovers in approximately 200   s . The recovery curve (dashed curve) is normalized to average intensity in a region far from the bleach; bleach in order to account for bleaching due to imaging. The scale bar in the image is 1 μm .

Fig. 3
Fig. 3

Examples of fitting experimental data. One-photon scheme: (a) Exemplary 3D one-photon bleach pattern obtained by scanning with v = 0.384 μm / s , Δ y = 0.0075 μm , n i t e r = 10 —a circular region with a radius of 0.1 μm with 1 .2 mW of the 488 nm line of an Ar laser. The color scale represents the fluorescence intensity range. (b) and (c) Fitting radial f 0 ( r , 0 ) and axial f 0 ( 0 , z ) behavior to the exponential Gaussian model components gives K = 1.21 ± 0.03 , w r = ( 0.82 ± 0.02 ) μm , and w z = ( 1.62 ± 0.08 ) μm . (d) The global fit is then superimposed on the experimental data contour plot, and the deviation between the data and the model is expressed in terms of the normalized sum of residuals S R = 0.078 . Two-photon scheme: (e) Exemplary 3D two-photon bleach pattern obtained by scanning with v = 0.384 μm / s , Δ y = 0.0075 μm , n i t e r = 12 a circular region with a radius of 0 .1 μm with 3 .8 mW at 790 nm . The color scale represents the fluorescence intensity range. (f) and (g) Fitting radial f 0 ( r , 0 ) and axial f 0 ( 0 , z ) behavior to the exponential Gaussian model components gives K = 0.89 ± 0.04 , w r = ( 0.91 ± 0.04 ) μm , and w z = ( 2.42 ± 0.16 ) μm . (h) The global fit is then superimposed on the experimental data contour plot, and the deviation between the data and the model is expressed in terms of the normalized sum of residuals S R = 0.045 .

Fig. 4
Fig. 4

On-axis components (radial and axial) of the experimental one-photon bleach profiles collected for different sizes of the bleaching region. All the profiles were obtained by scanning the circular regions with 1.1 mW of the 488 line of an Ar laser. The bleach scans were iterated ten times. The experimental profiles were fitted with the pure-radial and pure-axial parts of Eq. (9). The results of the fittingprocedure are shown in Table 1.

Fig. 5
Fig. 5

(a) Fit of experimental one-photon bleach profiles with the exponential-Gaussian approximation. The graphs show the results of the fit for experimental profiles obtained at different powers and and bleach phase times. By increasing bleach powers the w r and w z dimensions of the bleach profiles increase, indicating a fluorescence saturation effect. As expected, the increase in bleach power also increases K. Furthermore the sum of residuals also increases, showing that when using higher powers, deviation from the exponential-Gaussian approximation increases. (b) Fit of experimental two-photon bleach profiles with the exponential-Gaussian approximation. As in the case of the one-photon bleach profiles the increase of w r and w z depending on bleach power is indicative of a fluorescence saturation effect. However within the range of our measurements we did not notice an increase in the sum of residuals between the experimental profile and the fitted exponential Gaussian.

Fig. 6
Fig. 6

Maps of residuals between experimental bleach profiles and their best Gaussian-exponential approximation in the conventional [(a), (b), (c), and (d)] and the two-photon bleach regimes [(e), (f), (g), and (h)]. For direct comparison between confocal and two-photon situations the bleach parameter obtained from the fittingprocedure is indicated. In the one-photon regime when increasing bleaching time or bleaching power the model is unable to describe fluorescence intensity outside the optical axis and the focal plane. In the tested power range the model more closely approximates the profiles obtained in the two-photon regime.

Fig. 7
Fig. 7

(a) Fit of experimental two-photon bleach profiles obtained in a high-power bleach regime, with exponential-Gaussian approximation. Similar to the previous cases the increase of w r and w z depending on bleach power is representative of a fluorescence saturation effect. Within the range of our measurements we did not notice a significant increase in the sum of residuals between the experimental profile and the fitted exponential Gaussian. However the sums of residuals were higher than those in the previous two-photon measurements. (b) Maps of residuals between experimental bleach profiles and their best exponential-Gaussian approximation in high-energy two-photon bleach regime in two exemplary cases.

Fig. 8
Fig. 8

Results of the fitting of simulated FRAP data based on experimentally evaluated bleach profiles. The ratio between the estimated value of diffusion coefficient D fit and the true value D 0 is plotted against the bleach parameter K. When bleaching is performed in the one-photon setup (open symbols), the fit underestimates the diffusion coefficient. This underestimation is more pronounced when the bleach parameter K is higher. In the two-photon regime (filled symbols) the fit produced estimation of the diffusion coefficient within 20 % of the true value in the entire range of tested power levels. The different symbols show different acquisition conditions. More precisely the open circles and squares are the data obtained, respectively, by bleaching ten bleach scans for 2 ms in confocal setup. The filled squares and circles represent, respectively, data for 5 ms and 15 two-photon bleach scans with the first experimental setup. The black triangles represent of 15 two-photon bleach scans with the second experimental setup.

Tables (1)

Tables Icon

Table 1 Exponential-Gaussian Fit Results of the Radial and Axial One-Photon Bleaching Profiles for Bleaching Regions of Different Sizes w s

Equations (12)

Equations on this page are rendered with MathJax. Learn more.

d f ( x , y , z , t ) d t = k b I b l ( x , y , z ) f ( x , y , z , t ) ,
d f ( x , y , z , t ) d t = k 2 p h I b l 2 ( x , y , z ) f ( x , y , z , t ) ,
d f ( x , y , z , t ) d t = k b B ( x ( t ) , y ( t ) ) I b l ( x x ( t ) , y y ( t ) , z ) × f ( r , z , t ) .
f 0 ( x , y , z ) = f i n i ( x , y , z ) exp ( k b v Δ y B ( x , y ) I b l ( x , y , z ) ) ,
B ( x , y ) I b l ( x , y , z ) = + + B ( x , y ) × I b l ( x x , y y , z ) d x d y .
f 0 ( x , y , z ) = f i n i ( x , y , z ) exp ( k b v Δ y I b l ( x , y , z ) ) .
f 0 ( x , y , z ) = f i n i ( x , y , z ) exp ( k 2 p h v Δ y I b l 2 ( r , z ) ) .
I b l ( r , z ) = I b l ( 0 , 0 ) exp ( 2 r 2 w r 2 2 z 2 w z 2 ) ,
f 0 ( r , z ) = f i n i exp ( K 1 p h exp ( 2 r 2 w r 2 2 z 2 w z 2 ) ) ,
f 0 ( r , z ) = f i n i exp ( K 2 p h exp ( 4 r 2 w r 2 4 z 2 w z 2 ) ) ,
S R = i , j = 1 N r , N z | f 0 , exp ( r i , z j ) f 0 , f i t ( r i , z j ) | N r N z ,
f ( r , z , t ) t = D 0 r , z 2 f ( r , z , t ) ,

Metrics