Polarized fluorescent nanospheres

Rafal Luchowski; Zygmunt Gryczynski; Zeno Földes-Papp; Aaron Chang; Julian Borejdo; Pabak Sarkar; Ignacy Gryczynski

doi:10.1364/OE.18.004289

Polarized fluorescent nanospheres

Rafal Luchowski, Zygmunt Gryczynski, Zeno Földes-Papp, Aaron Chang, Julian Borejdo, Pabak Sarkar, and Ignacy Gryczynski

Optics Express
Vol. 18,
Issue 5,
pp. 4289-4299
(2010)
•https://doi.org/10.1364/OE.18.004289

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Rafal Luchowski, Zygmunt Gryczynski, Zeno Földes-Papp, Aaron Chang, Julian Borejdo, Pabak Sarkar, and Ignacy Gryczynski, "Polarized fluorescent nanospheres," Opt. Express 18, 4289-4299 (2010)

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Related Topics
Table of Contents Category
- Medical Optics and Biotechnology
Optics & Photonics Topics
?

The topics in this list come from the OSA Optics and Photonics Topics applied to this article.
Previously assigned OCIS codes
- Spectroscopy, fluorescence and luminescence (170.6280)
- Polarization (260.5430)

About this Article
History
- Original Manuscript: December 15, 2009
- Revised Manuscript: February 4, 2010
- Manuscript Accepted: February 4, 2010
- Published: February 17, 2010
Virtual Issues
Virtual Journal for Biomedical Optics Vol. 5, Iss. 6

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Open Access

Abstract

Fluorescent beads (nanoparticles, nanospheres) are commonly used in fluorescence spectroscopy and microscopy. Due to the random distribution of dye and high dye to nanoparticle ratio, the fluorescence polarization observed from the beads is low. Therefore beads are not used for polarization study. We demonstrate that photoselective bleaching creates beads with highly polarized fluorescence. First, the beads were immobilized in a PVA polymer. Second, the beads-doped PVA film was exposed to the illumination within the dye absorption band. A progressive decrease of absorption was observed. Next, photophysical properties of photobleached and not bleached films dissolved in water were compared.

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References

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Publication

P. V. Jena, P. S. Shirude, B. Okumus, K. Laxmi-Reddy, F. Godde, I. Huc, S. Balasubramanian, and T. Ha, “G-Quadruplex DNA Bound by a Synthetic Ligand is Highly Dynamic,” J. Am. Chem. Soc. 131(35), 12522–12523 (2009).
[Crossref] [PubMed]
S. Hohng and T. Ha, “Single-molecule quantum-dot fluorescence resonance energy transfer,” ChemPhysChem 6(5), 956–960 (2005).
[Crossref] [PubMed]
S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]
D. Evanko, “Nature Milestones in Light Microscopy. Milestone 17. Single molecules in the dark,” Nature (October): (2009), doi:.
H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]
L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]
H. P. Lu, “Single-molecule protein interaction conformational dynamics,” Curr. Pharm. Biotechnol. 10(5), 522–531 (2009).
[Crossref] [PubMed]
J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422(6930), 399–404 (2003).
[Crossref] [PubMed]
S. Syed, G. E. Snyder, C. Franzini-Armstrong, P. R. Selvin, and Y. E. Goldman, “Adaptability of myosin V studied by simultaneous detection of position and orientation,” EMBO J. 25(9), 1795–1803 (2006).
[Crossref] [PubMed]
A. P. Bartko and R. M. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B 103(51), 11237–11241 (1999).
[Crossref]
M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[Crossref] [PubMed]
A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[Crossref] [PubMed]
S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Rotational motions of macro-molecules by single-molecule fluorescence microscopy,” Acc. Chem. Res. 38(7), 583–593 (2005).
[Crossref] [PubMed]
T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]
A. M. De Grand, S. J. Lomnes, D. S. Lee, M. Pietrzykowski, S. Ohnishi, T. G. Morgan, A. Gogbashian, R. G. Laurence, and J. V. Frangioni, “Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons,” J. Biomed. Opt. 11(1), 10 (2006).
[Crossref]
E. Meiss, H. Konno, G. Groth, and T. Hisabori, “Molecular processes of inhibition and stimulation of ATP synthase caused by the phytotoxin tentoxin,” J. Biol. Chem. 283(36), 24594–24553 (2008).
[Crossref] [PubMed]
R. Luchowski, P. Sarkar, S. Bharill, G. Laczko, J. Borejdo, Z. Gryczynski, and I. Gryczynski, “Fluorescence polarization standard for near infrared spectroscopy and microscopy,” Appl. Opt. 47(33), 6257–6265 (2008).
[Crossref] [PubMed]
B. Valeur, “Molecular Fluorescence: Principles And Applications,” 125–198 (2006).
J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 5 (2005).
[Crossref]
I. Gregor and J. Enderlein, “Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy,” Photochem. Photobiol. Sci. 6(1), 13–18 (2007).
[Crossref] [PubMed]
E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[Crossref]
D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in reacting system: measurements by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]
D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]
R. Luchowski, E. G. Matveeva, I. Gryczynski, E. A. Terpetschnig, L. Patsenker, G. Laczko, J. Borejdo, and Z. Gryczynski, “Single Molecule Studies of Multiple-Fluorophore Labeled Antibodies. Effect of Homo-FRET on the Number of Photons Available Before Photobleaching,” Curr. Pharm. Biotechnol. 9(5), 411–420 (2008).
[Crossref] [PubMed]
M. L. Barcellona, S. Gammon, T. Hazlett, M. A. Digman, and E. Gratton, “Polarized fluorescence Correlation spectroscopy of DNA-DAPI complexes,” Microsc. Res. Tech. 65(4-5), 205–217 (2004).
[Crossref]

2009 (4)

P. V. Jena, P. S. Shirude, B. Okumus, K. Laxmi-Reddy, F. Godde, I. Huc, S. Balasubramanian, and T. Ha, “G-Quadruplex DNA Bound by a Synthetic Ligand is Highly Dynamic,” J. Am. Chem. Soc. 131(35), 12522–12523 (2009).
[Crossref] [PubMed]

D. Evanko, “Nature Milestones in Light Microscopy. Milestone 17. Single molecules in the dark,” Nature (October): (2009), doi:.

H. P. Lu, “Single-molecule protein interaction conformational dynamics,” Curr. Pharm. Biotechnol. 10(5), 522–531 (2009).
[Crossref] [PubMed]

T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]

2008 (3)

E. Meiss, H. Konno, G. Groth, and T. Hisabori, “Molecular processes of inhibition and stimulation of ATP synthase caused by the phytotoxin tentoxin,” J. Biol. Chem. 283(36), 24594–24553 (2008).
[Crossref] [PubMed]

R. Luchowski, P. Sarkar, S. Bharill, G. Laczko, J. Borejdo, Z. Gryczynski, and I. Gryczynski, “Fluorescence polarization standard for near infrared spectroscopy and microscopy,” Appl. Opt. 47(33), 6257–6265 (2008).
[Crossref] [PubMed]

R. Luchowski, E. G. Matveeva, I. Gryczynski, E. A. Terpetschnig, L. Patsenker, G. Laczko, J. Borejdo, and Z. Gryczynski, “Single Molecule Studies of Multiple-Fluorophore Labeled Antibodies. Effect of Homo-FRET on the Number of Photons Available Before Photobleaching,” Curr. Pharm. Biotechnol. 9(5), 411–420 (2008).
[Crossref] [PubMed]

2007 (1)

I. Gregor and J. Enderlein, “Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy,” Photochem. Photobiol. Sci. 6(1), 13–18 (2007).
[Crossref] [PubMed]

2006 (2)

A. M. De Grand, S. J. Lomnes, D. S. Lee, M. Pietrzykowski, S. Ohnishi, T. G. Morgan, A. Gogbashian, R. G. Laurence, and J. V. Frangioni, “Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons,” J. Biomed. Opt. 11(1), 10 (2006).
[Crossref]

S. Syed, G. E. Snyder, C. Franzini-Armstrong, P. R. Selvin, and Y. E. Goldman, “Adaptability of myosin V studied by simultaneous detection of position and orientation,” EMBO J. 25(9), 1795–1803 (2006).
[Crossref] [PubMed]

2005 (3)

S. Hohng and T. Ha, “Single-molecule quantum-dot fluorescence resonance energy transfer,” ChemPhysChem 6(5), 956–960 (2005).
[Crossref] [PubMed]

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 5 (2005).
[Crossref]

S. A. Rosenberg, M. E. Quinlan, J. N. Forkey, and Y. E. Goldman, “Rotational motions of macro-molecules by single-molecule fluorescence microscopy,” Acc. Chem. Res. 38(7), 583–593 (2005).
[Crossref] [PubMed]

2004 (2)

M. L. Barcellona, S. Gammon, T. Hazlett, M. A. Digman, and E. Gratton, “Polarized fluorescence Correlation spectroscopy of DNA-DAPI complexes,” Microsc. Res. Tech. 65(4-5), 205–217 (2004).
[Crossref]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]

2003 (2)

J. N. Forkey, M. E. Quinlan, M. A. Shaw, J. E. T. Corrie, and Y. E. Goldman, “Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization,” Nature 422(6930), 399–404 (2003).
[Crossref] [PubMed]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science 300(5628), 2061–2065 (2003).
[Crossref] [PubMed]

1999 (2)

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

A. P. Bartko and R. M. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B 103(51), 11237–11241 (1999).
[Crossref]

1998 (1)

H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]

1997 (1)

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[Crossref] [PubMed]

1974 (2)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[Crossref]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

1972 (1)

D. Magde, E. L. Elson, and W. W. Webb, “Thermodynamic fluctuations in reacting system: measurements by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[Crossref]

Balasubramanian, S.

Barcellona, M. L.

Bartko, A. P.

A. P. Bartko and R. M. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B 103(51), 11237–11241 (1999).
[Crossref]

Bharill, S.

Borejdo, J.

Cordes, T.

T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]

Corrie, J. E. T.

De Grand, A. M.

Dickson, R. M.

A. P. Bartko and R. M. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B 103(51), 11237–11241 (1999).
[Crossref]

Digman, M. A.

Edman, L.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

Elson, E. L.

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[Crossref]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Enderlein, J.

I. Gregor and J. Enderlein, “Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy,” Photochem. Photobiol. Sci. 6(1), 13–18 (2007).
[Crossref] [PubMed]

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 5 (2005).
[Crossref]

Evanko, D.

D. Evanko, “Nature Milestones in Light Microscopy. Milestone 17. Single molecules in the dark,” Nature (October): (2009), doi:.

Földes-Papp, Z.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

Forkey, J. N.

Frangioni, J. V.

Franzini-Armstrong, C.

Gammon, S.

Godde, F.

Gogbashian, A.

Goldman, Y. E.

Gratton, E.

Gregor, I.

I. Gregor and J. Enderlein, “Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy,” Photochem. Photobiol. Sci. 6(1), 13–18 (2007).
[Crossref] [PubMed]

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 5 (2005).
[Crossref]

Groth, G.

Gryczynski, I.

Gryczynski, Z.

Ha, T.

S. Hohng and T. Ha, “Single-molecule quantum-dot fluorescence resonance energy transfer,” ChemPhysChem 6(5), 956–960 (2005).
[Crossref] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]

Hazlett, T.

Hisabori, T.

Hohng, S.

S. Hohng and T. Ha, “Single-molecule quantum-dot fluorescence resonance energy transfer,” ChemPhysChem 6(5), 956–960 (2005).
[Crossref] [PubMed]

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]

Huc, I.

Iwane, A. H.

Jena, P. V.

Joo, C.

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]

Kitamura, K.

Konno, H.

Laczko, G.

Laurence, R. G.

Laxmi-Reddy, K.

Lee, D. S.

Lomnes, S. J.

Lu, H. P.

H. P. Lu, “Single-molecule protein interaction conformational dynamics,” Curr. Pharm. Biotechnol. 10(5), 522–531 (2009).
[Crossref] [PubMed]

H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]

Luchowski, R.

Magde, D.

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[Crossref]

Matveeva, E. G.

McKinney, S. A.

Meiss, E.

Morgan, T. G.

Ohnishi, S.

Okumus, B.

Patsenker, L.

Pietrzykowski, M.

Quinlan, M. E.

Rigler, R.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

Rosenberg, S. A.

Saito, K.

Sarkar, P.

Selvin, P. R.

Shaw, M. A.

Shirude, P. S.

Snyder, G. E.

Syed, S.

Terpetschnig, E. A.

Tinnefeld, P.

T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]

Tokunaga, M.

Vogelsang, J.

T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]

Webb, W. W.

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Wennmalm, S.

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

Xie, X. S.

H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]

Xun, L.

H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]

Yanagida, T.

Yildiz, A.

Acc. Chem. Res. (1)

Appl. Opt. (1)

Biochem. Biophys. Res. Commun. (1)

Biophys. J. (1)

S. Hohng, C. Joo, and T. Ha, “Single-molecule three-color FRET,” Biophys. J. 87(2), 1328–1337 (2004).
[Crossref] [PubMed]

Biopolymers (2)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[Crossref]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[Crossref] [PubMed]

Chem. Phys. (1)

L. Edman, Z. Földes-Papp, S. Wennmalm, and R. Rigler, “The fluctuating enzyme: a single molecule approach,” Chem. Phys. 247(1), 11–22 (1999).
[Crossref]

ChemPhysChem (1)

S. Hohng and T. Ha, “Single-molecule quantum-dot fluorescence resonance energy transfer,” ChemPhysChem 6(5), 956–960 (2005).
[Crossref] [PubMed]

Curr. Pharm. Biotechnol. (2)

H. P. Lu, “Single-molecule protein interaction conformational dynamics,” Curr. Pharm. Biotechnol. 10(5), 522–531 (2009).
[Crossref] [PubMed]

EMBO J. (1)

J. Am. Chem. Soc. (2)

T. Cordes, J. Vogelsang, and P. Tinnefeld, “On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent,” J. Am. Chem. Soc. 131, 5018 (2009).
[Crossref] [PubMed]

J. Biol. Chem. (1)

J. Biomed. Opt. (1)

J. Phys. Chem. B (1)

A. P. Bartko and R. M. Dickson, “Imaging three-dimensional single molecule orientations,” J. Phys. Chem. B 103(51), 11237–11241 (1999).
[Crossref]

Microsc. Res. Tech. (1)

Nature (2)

D. Evanko, “Nature Milestones in Light Microscopy. Milestone 17. Single molecules in the dark,” Nature (October): (2009), doi:.

Photochem. Photobiol. Sci. (1)

I. Gregor and J. Enderlein, “Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy,” Photochem. Photobiol. Sci. 6(1), 13–18 (2007).
[Crossref] [PubMed]

Phys. Rev. Lett. (1)

Rev. Sci. Instrum. (1)

J. Enderlein and I. Gregor, “Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy,” Rev. Sci. Instrum. 76(3), 5 (2005).
[Crossref]

Science (2)

H. P. Lu, L. Xun, and X. S. Xie, “Single-molecule enzymatic dynamics,” Science 282(5395), 1877–1882 (1998).
[Crossref] [PubMed]

Other (1)

B. Valeur, “Molecular Fluorescence: Principles And Applications,” 125–198 (2006).

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Fig. 1

Absorption spectra recorded for 24nm beads before (continuous) and after (dashed) progressive bleaching. After 8 hours photobleaching, the absorption decreased about 20 fold.

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Fig. 2

Emission spectra recorded for unbleached (top) and bleached (bottom) fluorescent nanospheres using 635nm excitation wavelength (continuous lines). Squares (top) and dots (bottom) present anisotropy data. The steady-state anisotropy is significantly higher for bleached nanospheres.

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Fig. 3

Schematic diagram for formation of the oriented transition dipole moment distribution in the fluorescence nanospheres (formation of polarized fluorescent nanospheres).

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Fig. 4

Anisotropy decays of the bleached and unbleached nanospheres. Bottom panels show residuals for the least square fits. The goodness of fit is reflected by the value of χ² given above. Excitation was 635nm, and emission was observed at 685nm with a long wave pass (LWP) filter >665nm.

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Fig. 5

Intensity decays of fluorescence of unbleached (a) and bleached (b) nanospheres. Black line: best two exponent fits. Bottom panels: residuals. Data collected using 635nm excitation from a pulsed solid state laser, emission was observed at 685nm with LWP665 filter.

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Fig. 6

Fluctuation number analysis of the measured fluorescence time traces in unbleached and bleached bead preparations. The total numbers of measured fluctuations above the background level of 5000Hz were in (a): 716, in (b): 210 and in (c): 514, in (d): 137. The measurement times were 600s. For preparative conditions see main text.

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Fig. 7

Auto- and crosscorrelation functions recorded for the unbleached (a) and bleached (b) nanospheres. Autocorrelation functions on both the pictures are presented with the same color label: green for autocorrelation signal from the detector recording fluorescence polarized perpendicular to the excitation and blue line for parallel to it. Crosscorrelation functions are presented with black dots together with fit marked in continuous red. Bottom panels of (a) and (b) presents residues.

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Tables (2)

Table 1 Fluorescence anisotropy decay parameters of the beads treated and not treated by light in aqueous solution (λ_exc. = 635nm, λ_obs. = 685nm).

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Table 2 Fluorescence lifetimes of the beads bleached and not bleached in aqueous solution measured under magic angle conditions (λ_exc.=635nm, λ_obs.=685nm).

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Equations (6)

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

I (t) = \sum_{i} α_{i} \exp (- t / τ_{i})

r (t) = \frac{I_{I I} (t) - G I_{⊥} (t)}{I_{I I} (t) + 2 G I_{⊥} (t)}

r (t) = \sum_{i = 1}^{n} R_{i} e^{- \frac{t}{φ_{i}}}

G_{k l} (τ) = \frac{〈 δ I_{k} (t) δ I_{l} (t + τ) 〉}{〈 δ I_{k} (t) 〉 〈 δ I_{l} (t + τ) 〉}

G (τ) = {\sum_{i = 1}^{n} ρ_{i} (1 + \frac{τ}{τ_{D i}})}^{- 1} {(1 + \frac{τ}{τ_{D i} κ^{2}})}^{- 1 / 2}

D = \frac{w_{o}^{2}}{4 τ_{D}}

Sample	R₁	Φ₁ [ns]	R₂	Φ₂ [ns]	Χ_R²
Not bleached nanospheres	0.15	0.35	0.05	4.46	0.83
Bleached nanospheres	0.06	0.31	0.30	140.1	1.34
$r (t) = \sum_{i = 1}^{n} R_{i} e^{- \frac{t}{φ_{i}}}$ , where θ_i are correlation times and R_i are associated amplitudes (anisotropies).

Sample	α₁	τ₁ [ns]	α ₂	τ ₂ [ns]	$\bar{τ}$ ^a [ns]	$〈 τ 〉$ ^b[ns]	Χ_R²
Not bleached nanospheres	0.53	2.74	0.47	4.83	4.02	3.73	0.98
Bleached nanospheres	0.2	1.1	0.8	5.03	4.83	4.26	0.93
^a $\bar{τ} = \sum_{i} f_{i} τ_{i}$ , $f_{i} = \frac{α_{i} τ_{i}}{\sum_{i} α_{i} τ_{i}}$ ^b $〈 τ 〉 = \sum_{i} α_{i} τ_{i}$

Sample	R₁	Φ₁ [ns]	R₂	Φ₂ [ns]	Χ_R²
Not bleached nanospheres	0.15	0.35	0.05	4.46	0.83
Bleached nanospheres	0.06	0.31	0.30	140.1	1.34
$r (t) = \sum_{i = 1}^{n} R_{i} e^{- \frac{t}{φ_{i}}}$ , where θ_i are correlation times and R_i are associated amplitudes (anisotropies).

Sample	α₁	τ₁ [ns]	α ₂	τ ₂ [ns]	$\bar{τ}$ ^a [ns]	$〈 τ 〉$ ^b[ns]	Χ_R²
Not bleached nanospheres	0.53	2.74	0.47	4.83	4.02	3.73	0.98
Bleached nanospheres	0.2	1.1	0.8	5.03	4.83	4.26	0.93
^a $\bar{τ} = \sum_{i} f_{i} τ_{i}$ , $f_{i} = \frac{α_{i} τ_{i}}{\sum_{i} α_{i} τ_{i}}$ ^b $〈 τ 〉 = \sum_{i} α_{i} τ_{i}$