Abstract

Discovery of Green Fluorescent Protein (GFP) constituted an important improvement for living cell studies on submicron resolution allowing in vivo fluorescence labeling. We studied the photo-physical properties of single GFP molecules incorporated in a charged polyelectrolyte environment by means of single molecule spectroscopy. The fluorescence characteristics change dramatically in terms of photo-stability, lifetime and blinking behavior so that the proteins scale up to quantum dots. The reported results highlight interesting applications in the design of fluorescent markers and in the development of optical data storage architectures.

© 2006 Optical Society of America

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References

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  1. O. Shimomura, “The discovery of aequorin and green fluorescent protein,” J. Microscopy 217, 3–15 (2005).
    [Crossref]
  2. A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
    [Crossref]
  3. 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]
  4. M. Zimmer, “Green fluorescent protein, Application structure and related photophysical behaviour.” Chem Rev. 102, 759–782 (2002).
    [Crossref] [PubMed]
  5. G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
    [Crossref]
  6. G.H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells.” Science. 297, 1873–1877 (2002).
    [Crossref] [PubMed]
  7. M. Schneider, S. Barozzi, I. Testa, M. Faretta, A. Diaspro, and A., “Two-photon activation and excitation properties of PA-GFP in the 720–920-nm region.” Biophys J. 89, 1346–1352 (2005).
    [Crossref] [PubMed]
  8. M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
    [Crossref]
  9. A. Zumbusch and G. Jung “Single Molecule Spectroscopy of the GFP, A Critical Assessment.” Single Mol. 1, 261–270 (2000).
    [Crossref]
  10. G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
    [Crossref] [PubMed]
  11. B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
    [Crossref] [PubMed]
  12. A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
    [Crossref]
  13. M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
    [Crossref] [PubMed]
  14. G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
    [Crossref]
  15. L.A. Deschenes and D.A. Vanden Bout, “Single molecule photobleaching, increasing photon yield and survival time through suppression of two-step photolysis.” Chem. Phys. Lett. 365, 387–395 (2002).
    [Crossref]
  16. W. van Sark et al. “Photooxidation and Photobleaching of Single CdSe/ZnS QDs probed by Room-Temperature Time-Resolved Spectroscopy.” J. Phys. Chem. B. 105, 8281–8284 (2001).
    [Crossref]
  17. G. Decher, “Fuzzy nanoassemblies, Toward layered polymeric multicomposites.” Science. 277, 1232–1237 (1997).
    [Crossref]
  18. E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).
  19. A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
    [Crossref]
  20. G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
    [Crossref]
  21. G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
    [Crossref] [PubMed]
  22. A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy.” Q. Rev. Biophys. 38(2), 97–166 (2005).
    [Crossref]
  23. F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
    [Crossref]
  24. J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
    [Crossref]
  25. G.H. Patterson and D.W. Piston, “Photobleaching in two-photon excitation microscopy.” Biophys. J. 78, 2159–2162 (2000).
    [Crossref] [PubMed]
  26. G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
    [Crossref] [PubMed]
  27. M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
    [Crossref] [PubMed]
  28. H.M. Berman et al., “The Protein Data Bank” Nucleic Acids Research 28, 235–242 (2000).
    [Crossref]
  29. The stabilizing and protecting effect in the polyelectrolyte matrix was also supported by chemically induced denaturation experiments. For this purpose, urea and guanidine hydrochloride were added to the proteins embedded in the polyelectrolyte layers and the disappearance of the fluorescence signals with time was recorded. For both agents a prolongation of the protein unfolding in comparison to wet silica gel enveloped GFPmut2 was found (data not shown).

2005 (8)

O. Shimomura, “The discovery of aequorin and green fluorescent protein,” J. Microscopy 217, 3–15 (2005).
[Crossref]

G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
[Crossref]

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

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy.” Q. Rev. Biophys. 38(2), 97–166 (2005).
[Crossref]

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

2004 (2)

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

2003 (1)

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

2002 (6)

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

L.A. Deschenes and D.A. Vanden Bout, “Single molecule photobleaching, increasing photon yield and survival time through suppression of two-step photolysis.” Chem. Phys. Lett. 365, 387–395 (2002).
[Crossref]

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

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

M. Zimmer, “Green fluorescent protein, Application structure and related photophysical behaviour.” Chem Rev. 102, 759–782 (2002).
[Crossref] [PubMed]

2001 (3)

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]

W. van Sark et al. “Photooxidation and Photobleaching of Single CdSe/ZnS QDs probed by Room-Temperature Time-Resolved Spectroscopy.” J. Phys. Chem. B. 105, 8281–8284 (2001).
[Crossref]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

2000 (4)

H.M. Berman et al., “The Protein Data Bank” Nucleic Acids Research 28, 235–242 (2000).
[Crossref]

G.H. Patterson and D.W. Piston, “Photobleaching in two-photon excitation microscopy.” Biophys. J. 78, 2159–2162 (2000).
[Crossref] [PubMed]

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

A. Zumbusch and G. Jung “Single Molecule Spectroscopy of the GFP, A Critical Assessment.” Single Mol. 1, 261–270 (2000).
[Crossref]

1997 (1)

G. Decher, “Fuzzy nanoassemblies, Toward layered polymeric multicomposites.” Science. 277, 1232–1237 (1997).
[Crossref]

1996 (1)

B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
[Crossref] [PubMed]

1995 (1)

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

A,

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

Adams, S.R.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

Andresen, M.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Baldini, G.

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Barozzi, S.

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

Beltram, F.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

Beretta, S.

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Berman, H.M.

H.M. Berman et al., “The Protein Data Bank” Nucleic Acids Research 28, 235–242 (2000).
[Crossref]

Bettati, S.

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

Bologna, S.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

Boyd, A.E.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

Caccia, M.

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

Campanini, B.

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

Cannone, F.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Caruso, F.

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Cavalleri, O.

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

Chanana, M.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Chirico, G.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy.” Q. Rev. Biophys. 38(2), 97–166 (2005).
[Crossref]

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Chodnevskaja, I.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Collini, M.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

A. Diaspro, G. Chirico, and M. Collini, “Two-photon fluorescence excitation and related techniques in biological microscopy.” Q. Rev. Biophys. 38(2), 97–166 (2005).
[Crossref]

Cormack, B.B.

B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
[Crossref] [PubMed]

Cubitt, A.B.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

Davis, S.A.

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Decher, G.

G. Decher, “Fuzzy nanoassemblies, Toward layered polymeric multicomposites.” Science. 277, 1232–1237 (1997).
[Crossref]

Deschenes, L.A.

L.A. Deschenes and D.A. Vanden Bout, “Single molecule photobleaching, increasing photon yield and survival time through suppression of two-step photolysis.” Chem. Phys. Lett. 365, 387–395 (2002).
[Crossref]

Diaspro, A.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

M. Schneider, S. Barozzi, I. Testa, M. Faretta, A. Diaspro, and A., “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(2), 97–166 (2005).
[Crossref]

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Donath, E.

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Doose, S.

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

Eggeling, C.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Falkow, S.

B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
[Crossref] [PubMed]

Faretta, M.

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

Galla, H.J.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Gliozzi, A.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

Gräber, F.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Gross, L.A.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

Grubmüller, H.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Heim, R.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

Hell, S.W.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Huewel, S.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Jakobs, S.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Jung, G.

G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
[Crossref]

A. Zumbusch and G. Jung “Single Molecule Spectroscopy of the GFP, A Critical Assessment.” Single Mol. 1, 261–270 (2000).
[Crossref]

Kallio, K.

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

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]

Krol, S.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

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]

Malengo, G.

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

Milani, R.

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

Mohwald, H.

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Moskalenko, V.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Mozzarelli, A.

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

Ormo, M.

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[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]

G.H. Patterson and D.W. Piston, “Photobleaching in two-photon excitation microscopy.” Biophys. J. 78, 2159–2162 (2000).
[Crossref] [PubMed]

Pellegrini, V.

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

Pinaud, F.

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

Piston, D.W.

G.H. Patterson and D.W. Piston, “Photobleaching in two-photon excitation microscopy.” Biophys. J. 78, 2159–2162 (2000).
[Crossref] [PubMed]

Remington, S.J.

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

Ruotolo, R.

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

S.J.,

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

Schäfer, L.V.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Schneider, M.

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

Shimomura, O.

O. Shimomura, “The discovery of aequorin and green fluorescent protein,” J. Microscopy 217, 3–15 (2005).
[Crossref]

Silvano, D.

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

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]

Stiel, A.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Sukhorukov, G.B.

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Testa, I.

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

Trowitzsch, S.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Tsay, J.M.

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

Tsien, R.Y.

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

Ulrichs, K.

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[Crossref] [PubMed]

Valdivia, R.H.

B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
[Crossref] [PubMed]

van Sark, W.

W. van Sark et al. “Photooxidation and Photobleaching of Single CdSe/ZnS QDs probed by Room-Temperature Time-Resolved Spectroscopy.” J. Phys. Chem. B. 105, 8281–8284 (2001).
[Crossref]

Vanden Bout, D.A.

L.A. Deschenes and D.A. Vanden Bout, “Single molecule photobleaching, increasing photon yield and survival time through suppression of two-step photolysis.” Chem. Phys. Lett. 365, 387–395 (2002).
[Crossref]

Wahl, M.C.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Weber, G.

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Weiss, S.

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

Wiehler, J.

G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
[Crossref]

Zimmer, M.

M. Zimmer, “Green fluorescent protein, Application structure and related photophysical behaviour.” Chem Rev. 102, 759–782 (2002).
[Crossref] [PubMed]

Zumbusch, A.

G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
[Crossref]

A. Zumbusch and G. Jung “Single Molecule Spectroscopy of the GFP, A Critical Assessment.” Single Mol. 1, 261–270 (2000).
[Crossref]

Angew.Che.Int.Ed. (1)

E. Donath, G.B. Sukhorukov, F. Caruso, S.A. Davis, and H. Mohwald, “Novel hollow polymer shells by colloid-templeted assembly of polyelectrolytes” Angew.Che.Int.Ed. 16, 2202–2205 (2000).

Biophys J. (1)

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

Biophys. J. (3)

G. Jung, J. Wiehler, and A. Zumbusch, “The Photophysics of GFP, influence of the Key Amino Acids at Positions 65 203 and 222.” Biophys. J. 88, 1932–1947 (2005).
[Crossref]

G.H. Patterson and D.W. Piston, “Photobleaching in two-photon excitation microscopy.” Biophys. J. 78, 2159–2162 (2000).
[Crossref] [PubMed]

G. Chirico, F. Cannone, G. Baldini, and A. Diaspro, “Two-photon thermal bleaching of single fluorescent molecules.” Biophys. J. 84, 588–598 (2003).
[Crossref] [PubMed]

Chem Rev. (1)

M. Zimmer, “Green fluorescent protein, Application structure and related photophysical behaviour.” Chem Rev. 102, 759–782 (2002).
[Crossref] [PubMed]

Chem. Phys. Chem. (1)

G. Chirico, A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram, “Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins induced by two-photon excitation.” Chem. Phys. Chem. 6, 328–335 (2005).
[Crossref] [PubMed]

Chem. Phys. Lett. (1)

L.A. Deschenes and D.A. Vanden Bout, “Single molecule photobleaching, increasing photon yield and survival time through suppression of two-step photolysis.” Chem. Phys. Lett. 365, 387–395 (2002).
[Crossref]

Gene. (1)

B.B. Cormack, R.H. Valdivia, and S. Falkow, “FACS-optimized mutants of green fluorescent protein (GFP).” Gene. 173, 33–38 (1996).
[Crossref] [PubMed]

IEEE Trans. Nanobiosci. (1)

A. Diaspro, S. Krol, O. Cavalleri, D. Silvano, and A. Gliozzi, “Microscopical Characterization of Nanocapsules Templated on Ionic Crystals and Biological Cells Toward Biomedical Applications.” IEEE Trans. Nanobiosci. 3, 110–115 (2002).
[Crossref]

J. Microscopy (1)

O. Shimomura, “The discovery of aequorin and green fluorescent protein,” J. Microscopy 217, 3–15 (2005).
[Crossref]

J. Phys. Chem. B. (2)

W. van Sark et al. “Photooxidation and Photobleaching of Single CdSe/ZnS QDs probed by Room-Temperature Time-Resolved Spectroscopy.” J. Phys. Chem. B. 105, 8281–8284 (2001).
[Crossref]

J.M. Tsay, S. Doose, F. Pinaud, and S. Weiss, “Enhancing the Photoluminescence of Peptide-Coated Nanocrystals with Shell Composition and UV Irradiation.” J. Phys. Chem. B. 109, 1669–1674 (2005).
[Crossref]

Langmuir. (1)

A. Diaspro, D. Silvano, S. Krol, O. Cavalleri, and A. Gliozzi, “Single living cell encapsulation in nano-organized polyelectrolyte shells.” Langmuir. 18, 5047–5050 (2002).
[Crossref]

Micr. Res Tech. (1)

G. Chirico, F. Cannone, S. Beretta, G. Baldini, and A. Diaspro, “Single Molecule Studies by Means of the Two-Photon Fluorescence Distribution.” Micr. Res Tech. 55, 359–364 (2001).
[Crossref]

Micr. Res. Tech. (1)

F. Cannone, M. Caccia, S. Bologna, A. Diaspro, and G. Chirico, “Single Molecule Spectroscopic Characterization of GFP-Mut2 Mutant for Two-Photon Microscopy Applications.” Micr. Res. Tech. 65, 186–193 (2004).
[Crossref]

Nano Lett.. (1)

M. Chanana, A. Gliozzi, A. Diaspro, I. Chodnevskaja, S. Huewel, V. Moskalenko, K. Ulrichs, H.J. Galla, and S. Krol, “Interaction of polyelectrolytes and their composites with living cells.” Nano Lett.. 5, 2605–2612 (2005).
[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]

Nucleic Acids Research (1)

H.M. Berman et al., “The Protein Data Bank” Nucleic Acids Research 28, 235–242 (2000).
[Crossref]

Proc. Natl. Acad. Sci. USA (1)

M. Andresen, M.C. Wahl, A. Stiel, F. Gräber, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, and S. Jakobs, “Structure and mechanism of the reversible photoswitch of a fluorescent protein.” Proc. Natl. Acad. Sci. USA 102 (37), 13070–13074 (2005).
[Crossref]

Protein Science. (1)

G. Chirico, F. Cannone, S. Beretta, A. Diaspro, B. Campanini, S. Bettati, R. Ruotolo, and A. Mozzarelli, “Dynamics of green GFPmut2 in solution on spin-coated glasses and encapsulated in wet silica gels.” Protein Science. 11, 1152–1161 (2002).
[Crossref] [PubMed]

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(2), 97–166 (2005).
[Crossref]

Rev. Sci. Instrum. (1)

G. Malengo, R. Milani, F. Cannone, S. Krol, A. Diaspro, and G. Chirico, “High sensitivity optical microscope for single molecule spectroscopy studies.” Rev. Sci. Instrum. 75, 2746–2751 (2004).
[Crossref]

Science. (2)

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

G. Decher, “Fuzzy nanoassemblies, Toward layered polymeric multicomposites.” Science. 277, 1232–1237 (1997).
[Crossref]

Single Mol. (1)

A. Zumbusch and G. Jung “Single Molecule Spectroscopy of the GFP, A Critical Assessment.” Single Mol. 1, 261–270 (2000).
[Crossref]

Trends in Biochem. Sci. (1)

A.B. Cubitt, R. Heim, S.R. Adams, A.E. Boyd, L.A. Gross, and R.Y. Tsien, “Understanding using and improving green fluorescent protein,” Trends in Biochem. Sci. 20, 448–455 (1995).
[Crossref]

Other (2)

The stabilizing and protecting effect in the polyelectrolyte matrix was also supported by chemically induced denaturation experiments. For this purpose, urea and guanidine hydrochloride were added to the proteins embedded in the polyelectrolyte layers and the disappearance of the fluorescence signals with time was recorded. For both agents a prolongation of the protein unfolding in comparison to wet silica gel enveloped GFPmut2 was found (data not shown).

M. Ormo, A.B. Cubitt, K. Kallio, L.A. Gross, R.Y. Tsien, S.J. Remington, and S.J. “Crystal structure of the Aequorea victoria green fluorescent protein.” Science273, 1392–1395 (1996).
[Crossref] [PubMed]

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Figures (5)

Fig. 1.
Fig. 1.

GFPmut2 configurations in the capsule A), B) and in spin coated polyelectrolyte layers (red for PSS, blu for PAH). See Methods.

Fig, 2.
Fig, 2.

(Left) GFPmut2 molecules (bright green spots) incorporated in a polyelectrolyte matrix onto amorphous calcium carbonate (dark sphere). The field of view is 3.6×3.6 µm (Central) shows the brightness and (Right) is the lifetime image of (Left) panel. So, the left picture is a fluorescence image where each pixel corresponds to the average photons counted per ms. The pixel size of the image of the left panel (50×50 nm) is different from that of the lifetime and brightness images (144×144 nm). This enhancement in terms of visualization results in a much blurred image on central and right panels [14].

Fig. 3.
Fig. 3.

Number of collected photons before bleaching versus the excitation intensity. A(2) outside attached to PSS layer (□); A(1) family (∙) and B family (O) in the layers; silica gel (S(3) in table 1) (⋄); agarose gel (▼) as in [9].

Fig. 4.
Fig. 4.

Fluorescence trace of proteins belonging to family A(1) as function of the excitation energy at 6.8 kW/cm2 (red), 9.5 kW/cm2 (blue), and 12 kW/cm2 (green). Enlargement of the red trace shows the short switch-off time, inset (A). The frequency of blinking in dependence of the intensity reveals very low values, inset (B). The red solid line is the linear fit: ν=A+B [Intensity] of the data with the slope B=2.3±0.5 cm2 Hz/kW.

Fig. 5.
Fig. 5.

Proposed model for the GFP interaction with PSS polyelectrolyte [27]. (a) (deepview of Swiss-PDBview) [29]. The positively charge amino acids (red) are on a rim around the fluorophore (yellow). (b) Sketch of GFP charge distribution. (c) Sketch of GFP in a PSS (blue) charged environment. A rim of positive charges near the fluorophore induces a tightening and stiffening of the beta barrel, a better protection and an increased time before bleaching. (Sketch originally elaborated by S.Krol).

Tables (1)

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Table 1. Fluorescence parameters for different configurations. a

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