Abstract

In this work we systematically explored performance of an EM-CCD as a detector for spatially resolved total internal reflection image correlation spectroscopy (TIR-ICS) with respect to adjustable parameters. We show that variations in the observation volume (pixel binning) can be well described by a simple structural term ω. To test the sensitivity of camera-based TIR-ICS we measured diffusion coefficients and particle numbers (PN) of fluorescent probes of different sizes (Fluorospheres, GFP and labeled antibodies) at varying viscosities, concentrations, and sampling rates. TIR-ICS allowed distinguishing between different probe concentrations with differences in PN of 5% and differences of 6% in D by acquiring only 15 independent measurement runs.

©2010 Optical Society of America

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    [Crossref] [PubMed]
  3. N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
    [Crossref] [PubMed]
  4. D Madge, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
    [Crossref]
  5. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
    [Crossref] [PubMed]
  6. M. Eigen, “Prionics or the kinetic basis of prion diseases,” Biophys. Chem. 63(1), A1–A18 (1996).
    [Crossref] [PubMed]
  7. J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
    [Crossref]
  8. T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys. J. 80(3), 1575–1584 (2001).
    [Crossref] [PubMed]
  9. K. K. Kuricheti, V. Buschmann, and K. D. Weston, “Application of fluorescence correlation spectroscopy for velocity imaging in microfluidic devices,” Appl. Spectrosc. 58(10), 1180–1186 (2004).
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    [Crossref] [PubMed]
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  13. P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
    [Crossref] [PubMed]
  14. B. C. Lagerholm and N. L. Thompson, “Theory for ligand rebinding at cell membrane surfaces,” Biophys. J. 74(3), 1215–1228 (1998).
    [Crossref] [PubMed]
  15. C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref]
  21. B. R. Terry, E. K. Matthews, and J. Haseloff, “Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy,” Biochem. Biophys. Res. Commun. 217(1), 21–27 (1995).
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    [Crossref] [PubMed]
  23. S. E. Sund, J. A. Swanson, and D. Axelrod, “Cell membrane orientation visualized by polarized total internal reflection fluorescence,” Biophys. J. 77(4), 2266–2283 (1999).
    [Crossref] [PubMed]
  24. D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
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2008 (2)

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[Crossref]

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[Crossref] [PubMed]

2007 (6)

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

K. Bacia and P. Schwille, “Fluorescence correlation spectroscopy,” Methods Mol. Biol. 398, 73–84 (2007).
[Crossref]

S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007).
[Crossref] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
[Crossref] [PubMed]

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[Crossref] [PubMed]

2006 (2)

O. Kochubey, A. Majumdar, and J. Klingauf, “Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission,” Traffic 7(12), 1614–1627 (2006).
[Crossref] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

2005 (4)

K. Hassler, T. Anhut, R. Rigler, M. Goesch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J.  88(1), L01–3 (2005).
[Crossref]

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and T. Lasser, “Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule,” Opt. Express 13(19), 7415–7423 (2005).
[Crossref] [PubMed]

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
[Crossref] [PubMed]

D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
[Crossref] [PubMed]

2004 (1)

2001 (1)

T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys. J. 80(3), 1575–1584 (2001).
[Crossref] [PubMed]

1999 (1)

S. E. Sund, J. A. Swanson, and D. Axelrod, “Cell membrane orientation visualized by polarized total internal reflection fluorescence,” Biophys. J. 77(4), 2266–2283 (1999).
[Crossref] [PubMed]

1998 (1)

B. C. Lagerholm and N. L. Thompson, “Theory for ligand rebinding at cell membrane surfaces,” Biophys. J. 74(3), 1215–1228 (1998).
[Crossref] [PubMed]

1997 (1)

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

1996 (1)

M. Eigen, “Prionics or the kinetic basis of prion diseases,” Biophys. Chem. 63(1), A1–A18 (1996).
[Crossref] [PubMed]

1995 (1)

B. R. Terry, E. K. Matthews, and J. Haseloff, “Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy,” Biochem. Biophys. Res. Commun. 217(1), 21–27 (1995).
[Crossref] [PubMed]

1974 (2)

D Madge, E. L. Elson, and W. W. Webb, “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]

Ahmed, S.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

Anhut, T.

K. Hassler, T. Anhut, R. Rigler, M. Goesch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J.  88(1), L01–3 (2005).
[Crossref]

Axelrod, D.

S. E. Sund, J. A. Swanson, and D. Axelrod, “Cell membrane orientation visualized by polarized total internal reflection fluorescence,” Biophys. J. 77(4), 2266–2283 (1999).
[Crossref] [PubMed]

Bacia, K.

K. Bacia and P. Schwille, “Fluorescence correlation spectroscopy,” Methods Mol. Biol. 398, 73–84 (2007).
[Crossref]

Buschmann, V.

Dertinger, T.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Ding, J. L.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

Eigen, M.

M. Eigen, “Prionics or the kinetic basis of prion diseases,” Biophys. Chem. 63(1), A1–A18 (1996).
[Crossref] [PubMed]

Elson, E. L.

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

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

Enderlein, J.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Goesch, M.

K. Hassler, T. Anhut, R. Rigler, M. Goesch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J.  88(1), L01–3 (2005).
[Crossref]

Gösch, M.

Gratton, E.

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[Crossref] [PubMed]

Gregor, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Guo, L.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

Har, J. Y.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Haseloff, J.

B. R. Terry, E. K. Matthews, and J. Haseloff, “Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy,” Biochem. Biophys. Res. Commun. 217(1), 21–27 (1995).
[Crossref] [PubMed]

Hassler, K.

Haustein, E.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[Crossref] [PubMed]

Heinze, K. G.

S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007).
[Crossref] [PubMed]

Kannan, B.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

Kim, S. A.

S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007).
[Crossref] [PubMed]

Klingauf, J.

O. Kochubey, A. Majumdar, and J. Klingauf, “Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission,” Traffic 7(12), 1614–1627 (2006).
[Crossref] [PubMed]

D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
[Crossref] [PubMed]

Kochubey, O.

O. Kochubey, A. Majumdar, and J. Klingauf, “Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission,” Traffic 7(12), 1614–1627 (2006).
[Crossref] [PubMed]

D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
[Crossref] [PubMed]

Kuricheti, K. K.

Lagerholm, B. C.

B. C. Lagerholm and N. L. Thompson, “Theory for ligand rebinding at cell membrane surfaces,” Biophys. J. 74(3), 1215–1228 (1998).
[Crossref] [PubMed]

Langowski, J.

J. Langowski, “Protein-protein interactions determined by fluorescence correlation spectroscopy,” Methods Cell Biol. 85, 471–484 (2008).
[Crossref]

Lasser, T.

Leutenegger, M.

Liu, P.

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

Loerke, D.

D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
[Crossref] [PubMed]

Madge, D

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

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]

Majumdar, A.

O. Kochubey, A. Majumdar, and J. Klingauf, “Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission,” Traffic 7(12), 1614–1627 (2006).
[Crossref] [PubMed]

Maruyama, I.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

Matthews, E. K.

B. R. Terry, E. K. Matthews, and J. Haseloff, “Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy,” Biochem. Biophys. Res. Commun. 217(1), 21–27 (1995).
[Crossref] [PubMed]

Merrifield, C. J.

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
[Crossref] [PubMed]

Meyer-Almes, F. J.

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Perrais, D.

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
[Crossref] [PubMed]

Rao, R.

Rigler, P.

Rigler, R.

K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and T. Lasser, “Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule,” Opt. Express 13(19), 7415–7423 (2005).
[Crossref] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Goesch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys J.  88(1), L01–3 (2005).
[Crossref]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Schwille, P.

E. Haustein and P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36(1), 151–169 (2007).
[Crossref] [PubMed]

S. A. Kim, K. G. Heinze, and P. Schwille, “Fluorescence correlation spectroscopy in living cells,” Nat. Methods 4(11), 963–973 (2007).
[Crossref] [PubMed]

K. Bacia and P. Schwille, “Fluorescence correlation spectroscopy,” Methods Mol. Biol. 398, 73–84 (2007).
[Crossref]

P. Schwille, F. J. Meyer-Almes, and R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution,” Biophys. J. 72(4), 1878–1886 (1997).
[Crossref] [PubMed]

Starr, T. E.

T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys. J. 80(3), 1575–1584 (2001).
[Crossref] [PubMed]

Steele, B. L.

N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
[Crossref] [PubMed]

Sudhaharan, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

Sund, S. E.

S. E. Sund, J. A. Swanson, and D. Axelrod, “Cell membrane orientation visualized by polarized total internal reflection fluorescence,” Biophys. J. 77(4), 2266–2283 (1999).
[Crossref] [PubMed]

Swanson, J. A.

S. E. Sund, J. A. Swanson, and D. Axelrod, “Cell membrane orientation visualized by polarized total internal reflection fluorescence,” Biophys. J. 77(4), 2266–2283 (1999).
[Crossref] [PubMed]

Terry, B. R.

B. R. Terry, E. K. Matthews, and J. Haseloff, “Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy,” Biochem. Biophys. Res. Commun. 217(1), 21–27 (1995).
[Crossref] [PubMed]

Thompson, N. L.

N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
[Crossref] [PubMed]

T. E. Starr and N. L. Thompson, “Total internal reflection with fluorescence correlation spectroscopy: combined surface reaction and solution diffusion,” Biophys. J. 80(3), 1575–1584 (2001).
[Crossref] [PubMed]

B. C. Lagerholm and N. L. Thompson, “Theory for ligand rebinding at cell membrane surfaces,” Biophys. J. 74(3), 1215–1228 (1998).
[Crossref] [PubMed]

Unruh, J. R.

J. R. Unruh and E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera,” Biophys. J. 95(11), 5385–5398 (2008).
[Crossref] [PubMed]

von der Hocht, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[Crossref] [PubMed]

Webb, W. W.

D Madge, E. L. Elson, and W. W. Webb, “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]

Weston,, K. D.

Wienisch, M.

D. Loerke, M. Wienisch, O. Kochubey, and J. Klingauf, “Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy,” Traffic 6(10), 918–929 (2005).
[Crossref] [PubMed]

Wohland, T.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
[Crossref] [PubMed]

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Zenisek, D.

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
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B. Kannan, J. Y. Har, P. Liu, I. Maruyama, J. L. Ding, and T. Wohland, “Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy,” Anal. Chem. 78(10), 3444–3451 (2006).
[Crossref] [PubMed]

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79(12), 4463–4470 (2007).
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C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell 121(4), 593–606 (2005).
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ChemPhysChem (1)

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
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A. Stroebel, O. Welzel, J. Kornhuber, and T. W. Groemer, “Background determination-based detection of scattered peaks,” Microsc. Res. Tech.; published online (2010).

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Figures (7)

Fig. 1
Fig. 1 Definition and characterization of the observation volume. (a) Image of an immobilized 43-nm bead on a glass cover slip surface. Pixel size of 160 nm. Color map in arbitrary units. (b) Diagram of the convolution of different binned areas with a Gaussian approximation of the PSF (solid lines) and its Gaussian approximation (dotted lines) as a function of distance. The convolution of binned area and PSF vary from 1x1 binning (black) to 16x16 binning (yellow). (c) Estimation of D for GFP (upper Graph) and fluorospheres (fs, lower Graph) for different lateral observation areas (binning) plotted to the x-axis. The corresponding evanescent field depth was held constant to 100 nm. (d) Approximation of the lateral part of the structure term, ωxy, by a linear function. The estimated values of ωxy (black) are plotted to the y-axis while the binning is shown on the x-axis. The measured values are fitted by a linear function (red line).

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Fig. 2
Fig. 2 Estimated diffusion coefficients for different evanescent field depths. The measured values are plotted with their standard deviation. The lateral observation volume was held constant to ωxy = 316.9 nm.

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Fig. 3
Fig. 3 Particle number estimation. (a) Histogram of camera readout noise. Shown are the results of 5 independent measurements. (b) Exemplar image for particle number estimation. Pixel size 160 nm. Color map in arbitrary units. A small area on the right side of the image is covered by an aperture to readout the dark counts of the camera. (c) Comparison of the measured (black) and calculated (red) particle numbers (PN) of 40 nm fluorosphores in 50% glycerol-water solution.(d) Dependency of the estimated number of particles in the whole 3D readout volume to changes in the lateral observation volume (black). Data could be fitted by a linear function (red line).

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Fig. 4
Fig. 4 Estimated diffusion coefficients of two different fluorescent particles, GFP (upper graph) and fluorospheres (lower graph) within different glycerol concentrations corresponding to varying viscosities. Data (black) were approximated by the Stokes-Einstein-Law (red fit). The diffusion coefficients of both traces are plotted to a linear scale of glycerol viscosity. All data were acquired with a camera speed of 1200 Hz and a binning of 1x1 (ωxy = 316.9 nm). The penetration depth varies, due to the different refractive indices of various glycerol solutions, from 89 nm to 256 nm (at a constant angle of incidence of 72°).

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Fig. 5
Fig. 5 Particle size alteration measurements. (a) Shown are autocorrelation traces from fluorescently labeled secondary IgG antibodies. The blue trace represents the ACF of fluorescently labeled secondary anti mouse antibodies. The red trace shows the ACF of secondary anti rabbit antibodies mixed with primary anti mouse IgG and the grey ACF originates from labeled anti mouse antibodies mixed with primary anti mouse antibodies. The blue and red curves are almost overlapping. (b) Comparison of estimated diffusion coefficients for IgG binding. Bars represent the diffusion coefficients of.labeled anti mouse antibodies mixed with primary anti mouse antibodies.(grey), secondary anti mouse antibodies (blue), and anti rabbit antibodies mixed with primary anti mouse IgG (red).

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Fig. 6
Fig. 6 Resolving power in particle number measurements of 40 nm beads in dependence of the total number of particles in the observation volume of 1.78 fl (16x binning, 84 nm penetration depth).

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Fig. 7
Fig. 7 Measurement of the resolving power for estimating diffusion coefficients. (a) Dependence of the minimal relative resolvable difference of diffusion coefficients, RRdDmin respectively (color bar) on the quotient of sampling time and lateral and axial diffusion times, ST/τxy and ST/τz respectively. The resolution of discriminating diffusion coefficients decreases from the blue to the red color. (b) Dependence of the minimal relative resolvable difference of diffusion coefficients (RRdDmin) on the total number of observation volume crossings for a sampling frequency of 1.2 kHz and diffusing GFP molecules in 80% glycerol. Data could be fitted by a hyperbolic function (red).

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Equations (14)

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θ = sin − 1 ( N A n o i l )
d = λ 4 π ( n g l a s s 2 ⋅ sin 2 ( θ ) − n s o l u t i o n 2 )
M D E ( q ) = ∫ D e t s i z e ( q ' ) ⋅ P S F ( q − q ' ) d q '
M D E ( x , y , z ) = exp ( − 2 x 2 + y 2 ω x y 2 ) · exp ( − z d )
W n = ∫ V M D E ( r ) n d r ,
V e f f = W 1 2 W 2
G ( τ ) = < δ F ( t ) ⋅ δ F ( t + τ ) > < F ( t ) > 2 ,
G ( τ ) = γ N ( 1 + τ ω 2 τ z ) − 1 [ ( 1 − τ 2 τ z ) w ( i τ 4 τ z ) + τ π τ z ]
w ( x ) = exp ( − x 2 ) e r f c ( − i x )
ω = ω x y d
τ z = d 2 4 D
N = α ⋅ γ ( < F ( t ) > − b a c k g r o u n d ) 2 < δ F ( t ) 2 >
ω x y = a ⋅ D e t S i z e + b
τ = ω x y d ⋅ τ x y + τ z ω x y d + 1

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