Abstract

Multi-modal nanoparticles incorporating fluorophores are increasingly being used for medical applications. The number of fluorophores incorporated into the nanoparticles during synthesis is stochastic, leaving some nanoparticles devoid of fluorophores. Determining the number, the brightness and the photostability of the fluorophores incorporated, and the percentage of labeled nanoparticles (labeling efficiency) remains challenging. We have determined the aforementioned quantities for two synthesized multi-modal nanoparticles by exploiting the photobleaching of fluorophores at the single-molecule level using a total internal reflection fluorescence microscope. Labeling efficiency was determined by fitting the distribution of incorporated fluorophores with a statistical model and verified by independent experiments.

© 2011 OSA

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2011

A. J. Cole, V. C. Yang, and A. E. David, “Cancer theranostics: the rise of targeted magnetic nanoparticles,” Trends Biotechnol.29(7), 323–332 (2011).
[CrossRef] [PubMed]

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

2010

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

2009

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

2008

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]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

S. Lal, S. E. Clare, and N. J. Halas, “Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc. Chem. Res.41(12), 1842–1851 (2008).
[CrossRef] [PubMed]

C. Y. Zhang and L. W. Johnson, “Simple and accurate quantification of quantum dots via single-particle counting,” J. Am. Chem. Soc.130(12), 3750–3751 (2008).
[CrossRef] [PubMed]

2007

M. H. Ulbrich and E. Y. Isacoff, “Subunit counting in membrane-bound proteins,” Nat. Methods4(4), 319–321 (2007).
[PubMed]

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

2006

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

2005

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

2004

L. Brannon-Peppas and J. O. Blanchette, “Nanoparticle and targeted systems for cancer therapy,” Adv. Drug Deliv. Rev.56(11), 1649–1659 (2004).
[CrossRef] [PubMed]

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

2001

R. Weissleder, “A clearer vision for in vivo imaging,” Nat. Biotechnol.19(4), 316–317 (2001).
[CrossRef] [PubMed]

1999

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science283(5408), 1676–1683 (1999).
[CrossRef] [PubMed]

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

1991

B. Efron and R. Tibshirani, “Statistical data analysis in the computer age,” Science253(5018), 390–395 (1991).
[CrossRef] [PubMed]

1977

A. H. Herz, “Aggregation of sensitizing dyes in solution and their adsorption onto silver-halides,” Adv. Colloid Interface Sci.8(4), 237–298 (1977).
[CrossRef]

Alexandrou, A.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Armitage, J. P.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Aslan, K.

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

Bai, F.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Baumgart, M.

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Benezra, M.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Bernardo, M.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

Berry, R. M.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Blanchette, J. O.

L. Brannon-Peppas and J. O. Blanchette, “Nanoparticle and targeted systems for cancer therapy,” Adv. Drug Deliv. Rev.56(11), 1649–1659 (2004).
[CrossRef] [PubMed]

Boilot, J.-P.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Borejdo, J.

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]

Bradbury, M.

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Bradbury, M. S.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Brannon-Peppas, L.

L. Brannon-Peppas and J. O. Blanchette, “Nanoparticle and targeted systems for cancer therapy,” Adv. Drug Deliv. Rev.56(11), 1649–1659 (2004).
[CrossRef] [PubMed]

Brechbiel, M. W.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Bumb, A.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Burns, A.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Burns, A. A.

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Casanova, D.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Chandler, J. H.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Chen, Z.

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

Choyke, P. L.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Clare, S. E.

S. Lal, S. E. Clare, and N. J. Halas, “Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc. Chem. Res.41(12), 1842–1851 (2008).
[CrossRef] [PubMed]

Cole, A. J.

A. J. Cole, V. C. Yang, and A. E. David, “Cancer theranostics: the rise of targeted magnetic nanoparticles,” Trends Biotechnol.29(7), 323–332 (2011).
[CrossRef] [PubMed]

Craighead, H. G.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

David, A. E.

A. J. Cole, V. C. Yang, and A. E. David, “Cancer theranostics: the rise of targeted magnetic nanoparticles,” Trends Biotechnol.29(7), 323–332 (2011).
[CrossRef] [PubMed]

de Lang, D. T. N.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Delport, F.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Deres, A.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

DeStanchina, E.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Dobson, P. J.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Edel, J. B.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

Efron, B.

B. Efron and R. Tibshirani, “Statistical data analysis in the computer age,” Science253(5018), 390–395 (1991).
[CrossRef] [PubMed]

Eggeman, A.

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Engelberts, J. J.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Fugger, L.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Gacoin, T.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Geddes, C. D.

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

Giaume, D.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Gryczynski, I.

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]

Gryczynski, Z.

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]

Halas, N. J.

S. Lal, S. E. Clare, and N. J. Halas, “Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc. Chem. Res.41(12), 1842–1851 (2008).
[CrossRef] [PubMed]

Herz, A. H.

A. H. Herz, “Aggregation of sensitizing dyes in solution and their adsorption onto silver-halides,” Adv. Colloid Interface Sci.8(4), 237–298 (1977).
[CrossRef]

Herz, E.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Hofkens, J.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Hotta, J.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Hutchinson, J.

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Imhof, A.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Isacoff, E. Y.

M. H. Ulbrich and E. Y. Isacoff, “Subunit counting in membrane-bound proteins,” Nat. Methods4(4), 319–321 (2007).
[PubMed]

Iyer, S.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Johnson, L. W.

C. Y. Zhang and L. W. Johnson, “Simple and accurate quantification of quantum dots via single-particle counting,” J. Am. Chem. Soc.130(12), 3750–3751 (2008).
[CrossRef] [PubMed]

Laczko, G.

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]

Lakowicz, J. R.

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

Lal, S.

S. Lal, S. E. Clare, and N. J. Halas, “Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc. Chem. Res.41(12), 1842–1851 (2008).
[CrossRef] [PubMed]

Lammertyn, J.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Larson, S. M.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Leake, M. C.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Lohr, A.

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

Longo, V.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Luchowski, R.

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]

Martin, J.-L.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Mashanov, G. I.

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

Matveeva, E. G.

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]

Megens, M.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Molloy, J. E.

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

Moreau, M.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

Ogawa, M.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

Ow, H.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Patsenker, L.

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]

Peckham, M.

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

Penate-Medina, O.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Perkins, M. R.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

Pollet, J.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Prabhakaran, D.

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Regino, C. A. S.

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

Saha-Möller, C. R.

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

Samiee, K. T.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

Schaer, D.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Sels, B.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Sprik, R.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Stavis, S. M.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

Tacon, D.

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

Terpetschnig, E. A.

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]

Tibshirani, R.

B. Efron and R. Tibshirani, “Statistical data analysis in the computer age,” Science253(5018), 390–395 (1991).
[CrossRef] [PubMed]

Ulbrich, M. H.

M. H. Ulbrich and E. Y. Isacoff, “Subunit counting in membrane-bound proteins,” Nat. Methods4(4), 319–321 (2007).
[PubMed]

Verbruggen, B.

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Vider, J.

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Vos, W. L.

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Wadhams, G. H.

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Weiss, S.

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science283(5408), 1676–1683 (1999).
[CrossRef] [PubMed]

Weissleder, R.

R. Weissleder, “A clearer vision for in vivo imaging,” Nat. Biotechnol.19(4), 316–317 (2001).
[CrossRef] [PubMed]

Wiesner, U.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Wolchok, J.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Wu, M.

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

Würthner, F.

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

Yang, V. C.

A. J. Cole, V. C. Yang, and A. E. David, “Cancer theranostics: the rise of targeted magnetic nanoparticles,” Trends Biotechnol.29(7), 323–332 (2011).
[CrossRef] [PubMed]

Zanzonico, P. B.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

Zhang, C. Y.

C. Y. Zhang and L. W. Johnson, “Simple and accurate quantification of quantum dots via single-particle counting,” J. Am. Chem. Soc.130(12), 3750–3751 (2008).
[CrossRef] [PubMed]

Acc. Chem. Res.

S. Lal, S. E. Clare, and N. J. Halas, “Nanoshell-enabled photothermal cancer therapy: impending clinical impact,” Acc. Chem. Res.41(12), 1842–1851 (2008).
[CrossRef] [PubMed]

Adv. Colloid Interface Sci.

A. H. Herz, “Aggregation of sensitizing dyes in solution and their adsorption onto silver-halides,” Adv. Colloid Interface Sci.8(4), 237–298 (1977).
[CrossRef]

Adv. Drug Deliv. Rev.

L. Brannon-Peppas and J. O. Blanchette, “Nanoparticle and targeted systems for cancer therapy,” Adv. Drug Deliv. Rev.56(11), 1649–1659 (2004).
[CrossRef] [PubMed]

Chem. Soc. Rev.

Z. Chen, A. Lohr, C. R. Saha-Möller, and F. Würthner, “Self-assembled pi-stacks of functional dyes in solution: structural and thermodynamic features,” Chem. Soc. Rev.38(2), 564–584 (2009).
[CrossRef] [PubMed]

Curr. Pharm. Biotechnol.

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]

J. Am. Chem. Soc.

D. Casanova, D. Giaume, M. Moreau, J.-L. Martin, T. Gacoin, J.-P. Boilot, and A. Alexandrou, “Counting the number of proteins coupled to single nanoparticles,” J. Am. Chem. Soc.129(42), 12592–12593 (2007).
[CrossRef] [PubMed]

C. Y. Zhang and L. W. Johnson, “Simple and accurate quantification of quantum dots via single-particle counting,” J. Am. Chem. Soc.130(12), 3750–3751 (2008).
[CrossRef] [PubMed]

K. Aslan, M. Wu, J. R. Lakowicz, and C. D. Geddes, “Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms,” J. Am. Chem. Soc.129(6), 1524–1525 (2007).
[CrossRef] [PubMed]

J. Biol. Chem.

G. I. Mashanov, D. Tacon, M. Peckham, and J. E. Molloy, “The spatial and temporal dynamics of pleckstrin homology domain binding at the plasma membrane measured by imaging single molecules in live mouse myoblasts,” J. Biol. Chem.279(15), 15274–15280 (2004).
[CrossRef] [PubMed]

J. Clin. Invest.

M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer, H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J. Wolchok, S. M. Larson, U. Wiesner, and M. S. Bradbury, “Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma,” J. Clin. Invest.121(7), 2768–2780 (2011).
[CrossRef] [PubMed]

J. Phys. Chem. B

A. Imhof, M. Megens, J. J. Engelberts, D. T. N. de Lang, R. Sprik, and W. L. Vos, “Spectroscopy of fluorescein (FITC) dyed colloidal silica spheres,” J. Phys. Chem. B103(9), 1408–1415 (1999).
[CrossRef]

Lab Chip

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, “Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel,” Lab Chip5(3), 337–343 (2005).
[CrossRef] [PubMed]

Langmuir

F. Delport, A. Deres, J. Hotta, J. Pollet, B. Verbruggen, B. Sels, J. Hofkens, and J. Lammertyn, “Improved method for counting DNA molecules on biofunctionalized nanoparticles,” Langmuir26(3), 1594–1597 (2010).
[CrossRef] [PubMed]

Nano Lett.

A. A. Burns, J. Vider, H. Ow, E. Herz, O. Penate-Medina, M. Baumgart, S. M. Larson, U. Wiesner, and M. Bradbury, “Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine,” Nano Lett.9(1), 442–448 (2009).
[CrossRef] [PubMed]

Nanotechnology

A. Bumb, C. A. S. Regino, M. R. Perkins, M. Bernardo, M. Ogawa, L. Fugger, P. L. Choyke, P. J. Dobson, and M. W. Brechbiel, “Preparation and characterization of a magnetic and optical dual-modality molecular probe,” Nanotechnology21(17), 175704 (2010).
[CrossRef] [PubMed]

A. Bumb, M. W. Brechbiel, P. L. Choyke, L. Fugger, A. Eggeman, D. Prabhakaran, J. Hutchinson, and P. J. Dobson, “Synthesis and characterization of ultra-small superparamagnetic iron oxide nanoparticles thinly coated with silica,” Nanotechnology19(33), 335601 (2008).
[CrossRef] [PubMed]

Nat. Biotechnol.

R. Weissleder, “A clearer vision for in vivo imaging,” Nat. Biotechnol.19(4), 316–317 (2001).
[CrossRef] [PubMed]

Nat. Methods

M. H. Ulbrich and E. Y. Isacoff, “Subunit counting in membrane-bound proteins,” Nat. Methods4(4), 319–321 (2007).
[PubMed]

Nature

M. C. Leake, J. H. Chandler, G. H. Wadhams, F. Bai, R. M. Berry, and J. P. Armitage, “Stoichiometry and turnover in single, functioning membrane protein complexes,” Nature443(7109), 355–358 (2006).
[CrossRef] [PubMed]

Science

S. Weiss, “Fluorescence spectroscopy of single biomolecules,” Science283(5408), 1676–1683 (1999).
[CrossRef] [PubMed]

B. Efron and R. Tibshirani, “Statistical data analysis in the computer age,” Science253(5018), 390–395 (1991).
[CrossRef] [PubMed]

Trends Biotechnol.

A. J. Cole, V. C. Yang, and A. E. David, “Cancer theranostics: the rise of targeted magnetic nanoparticles,” Trends Biotechnol.29(7), 323–332 (2011).
[CrossRef] [PubMed]

Other

A. Papoulis, Probability, Random Variables and Stochastic Processes (McGraw Hill Inc, New York, NY, 1991).

P. R. Selvin and T. Ha, Single Molecule Techniques: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Woodbury, NY, 2008).

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Figures (4)

Fig. 1
Fig. 1

Single Particle Observation Technique. (a) Dual-modal magnetic resonance/optical fluorescence silica-coated iron oxide nanoparticles (SCION) were dispersed in a sample chamber. (b) When incident light hits the boundary of two media at an angle greater than a critical angle, θc, total internal reflection occurs resulting in an evanescent field that excites fluorophores in a thin layer near the surface (~200nm). (c) Schematic of the TIRF microscope. The excitation laser (green) is refracted by the quartz prism and totally internally reflected at the quartz slide-sample interface. The fluorescence emission is collected by a microscope objective, filtered to remove the excitation light and imaged by an EMCCD camera.

Fig. 2
Fig. 2

Number of fluorophores incorporated in nanoparticles. (a) Fluorescence intensity as a function of time for a SCION(Alexa555) particle (red) and background (grey). Bleaching of individual fluorophores results in discrete decreases in intensity (black arrows). The number of bleaching events reveals the number of fluorophores in the nanoparticle. The bleaching times are indicated by dashed lines. (b) Distribution of the number of fluorophores for SCION(Cy5.5) (red circles). Fitting to a single fluorophore incorporation model (Eq. (1)), blue line, reduced χ2 = 3.3) returned an average of 1.58 ± 0.04 fluorophores per particle. Fitting to a fluorophore-cluster incorporation model (see text, red line, reduced χ2 = 1.6) returned an average of 0.78 ± 0.02 clusters per particle, with an average of 1.7 ± 0.1 fluorophores per cluster determined from the free fluorophore distribution (inset). (c) Distribution of the number of fluorophores in SCION(Alexa555) (red circles). Fitting to a single fluorophore incorporation model (Eq. (1)), blue line, reduced χ2 = 7.5) returned an average of 0.49 ± 0.02 fluorophores per particle. Fitting to a fluorophore-cluster incorporation model (see text, red line, reduced χ2 = 3.4) returned an average of 0.25 ± 0.02 clusters per particle, with an average of 1.15 ± 0.02 fluorophores per cluster determined from the free fluorophore distribution (inset). The uncertainties in the fit parameters were obtained by bootstrap analysis [24]. The free fluorophore cluster distributions were approximated by Poisson distributions (Eq. (1)), which were corrected to account for the fact that the clusters contain at least one fluorophore.

Fig. 3
Fig. 3

Experimental confirmation of particle labeling efficiency. (a) Spinning disk confocal fluorescence image gives the number of nanoparticles with fluorophores. (b) Phase contrast image of the same field of view gives the total number of nanoparticles with and without fluorophores. Every spot on the fluorescence image has a corresponding point in the phase contrast image (examples are encircled).

Fig. 4
Fig. 4

Photophysical properties of free and encapsulated Alexa555. (a) Distribution of fluorophore intensity drops for free fluorophore and (b) for SCION particles from individual intensity traces (Fig. 2a). (c) Fluorescence decays and fits for free fluorophore (black) and SCION(Alexa 555) particles (red). The fluorescence lifetimes were determined from the deconvolution fits. Free fluorophores had a single lifetime of 228 ± 2 ps, whereas encapsulated fluorophores had two lifetimes of 228 ± 2 ps and 1.5 ± 0.1 ns. (d) Distribution of fluorophore bleaching lifetimes obtained from individual intensity traces (Fig. 2a) of SCION(Alexa 555) particles (red circles) and free fluorophores (black circles) at 6 mW of laser power. The distributions were fit with single-exponential decays. The lifetime of encapsulated fluorophores was 89 ± 4 s (red line, reduced χ2 = 2.3), whereas the lifetime of free Alexa555 was 23 ± 2 s (black line, reduced χ2 = 0.85). (e) Bleaching time as a function of laser power for SCION(Alexa 555) particles (red) and free fluorophores (black).

Equations (5)

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P(n)= λ n e λ n!
P particle (c)= λ cluster c e λ cluster c! , c = 0, 1, 2, 3… 
P cluster (d)= λ dye d e λ dye d! , d = 1, 2, 3… 
Labeling  Efficiency= P particle (c1) =1 P particle (0) =1 e λ cluster
F(t)= F 0 e t τ 1 τ = k i

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