Abstract

Supercritical angle fluorescence (SAF) detection combines the axial discrimination and exquisite signal-to-noise ratio of total internal reflection fluorescence (TIRF) with the lateral discrimination and convenience of confocal excitation. This combination makes SAF ideal for fluorescence correlation spectroscopy (FCS) on membranes and other structures in close proximity to the coverslip. Here we report a straightforward modification of a commercial microscope to implement SAF FCS and demonstrate in both model supported lipid bilayers and cellular systems that this approach shows an increase in signal from membrane-bound fluorophores relative to fluorophores in solution, benchmarked against line-scanning FCS. SAF FCS allowed us to demonstrate that activation of the T cell receptor resulted in the recruitment of the kinase Lck to the plasma membrane as well as a reduction in Lck mobility within the membrane.

© 2016 Optical Society of America

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References

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  1. D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscopy,” Trends Cell Biol. 11(7), 298–303 (2001).
    [Crossref] [PubMed]
  2. T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12(18), 4246–4254 (2004).
    [Crossref] [PubMed]
  3. T. Ruckstuhl, D. Verdes, C. M. Winterflood, and S. Seeger, “Simultaneous near-field and far-field fluorescence microscopy of single molecules,” Opt. Express 19(7), 6836–6844 (2011).
    [Crossref] [PubMed]
  4. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008).
    [Crossref]
  5. J. Deschamps, M. Mund, and J. Ries, “3D superresolution microscopy by supercritical angle detection,” Opt. Express 22(23), 29081–29091 (2014).
    [Crossref] [PubMed]
  6. C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
    [Crossref] [PubMed]
  7. T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, “Full-field supercritical angle fluorescence microscopy for live cell imaging,” Opt. Lett. 36(16), 3051–3053 (2011).
    [Crossref] [PubMed]
  8. J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
    [Crossref] [PubMed]
  9. N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
    [Crossref] [PubMed]
  10. J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
    [Crossref] [PubMed]
  11. M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
    [Crossref] [PubMed]
  12. C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
    [Crossref] [PubMed]
  13. J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
    [Crossref] [PubMed]
  14. K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
    [Crossref] [PubMed]
  15. H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
    [Crossref] [PubMed]
  16. L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
    [Crossref] [PubMed]
  17. N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
    [Crossref]
  18. A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
    [Crossref] [PubMed]
  19. J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
    [Crossref] [PubMed]
  20. A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
    [Crossref]
  21. F. G. Gervais and A. Veillette, “The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes,” Mol. Cell. Biol. 15(5), 2393–2401 (1995).
    [Crossref] [PubMed]
  22. U. Mets and R. Rigler, “Submillisecond detection of single rhodamine molecules in water,” J. Fluoresc. 4(3), 259–264 (1994).
    [Crossref] [PubMed]
  23. J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
    [Crossref] [PubMed]
  24. E. Conibear and N. G. Davis, “Palmitoylation and depalmitoylation dynamics at a glance,” J. Cell Sci. 123(23), 4007–4010 (2010).
    [Crossref] [PubMed]
  25. A. D. Douglass and R. D. Vale, “Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells,” Cell 121(6), 937–950 (2005).
    [Crossref] [PubMed]

2015 (2)

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
[Crossref] [PubMed]

2014 (2)

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

J. Deschamps, M. Mund, and J. Ries, “3D superresolution microscopy by supercritical angle detection,” Opt. Express 22(23), 29081–29091 (2014).
[Crossref] [PubMed]

2012 (2)

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

2011 (2)

2010 (3)

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

E. Conibear and N. G. Davis, “Palmitoylation and depalmitoylation dynamics at a glance,” J. Cell Sci. 123(23), 4007–4010 (2010).
[Crossref] [PubMed]

2009 (1)

J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[Crossref] [PubMed]

2008 (4)

J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[Crossref] [PubMed]

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008).
[Crossref]

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
[Crossref] [PubMed]

2007 (1)

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

2005 (1)

A. D. Douglass and R. D. Vale, “Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells,” Cell 121(6), 937–950 (2005).
[Crossref] [PubMed]

2004 (1)

2003 (2)

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

2001 (1)

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscopy,” Trends Cell Biol. 11(7), 298–303 (2001).
[Crossref] [PubMed]

1995 (1)

F. G. Gervais and A. Veillette, “The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes,” Mol. Cell. Biol. 15(5), 2393–2401 (1995).
[Crossref] [PubMed]

1994 (1)

U. Mets and R. Rigler, “Submillisecond detection of single rhodamine molecules in water,” J. Fluoresc. 4(3), 259–264 (1994).
[Crossref] [PubMed]

1987 (1)

C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
[Crossref] [PubMed]

Acuto, O.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Balaa, K.

Barroca, T.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, “Full-field supercritical angle fluorescence microscopy for live cell imaging,” Opt. Lett. 36(16), 3051–3053 (2011).
[Crossref] [PubMed]

Benda, A.

A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
[Crossref] [PubMed]

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Beneš, M.

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Bettens, F.

C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
[Crossref] [PubMed]

Bista, M.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Bon, P.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Bourg, N.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Bradke, F.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Cerundolo, V.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Chiantia, S.

J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[Crossref] [PubMed]

Conibear, E.

E. Conibear and N. G. Davis, “Palmitoylation and depalmitoylation dynamics at a glance,” J. Cell Sci. 123(23), 4007–4010 (2010).
[Crossref] [PubMed]

Crevenna, A. H.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Davis, N. G.

E. Conibear and N. G. Davis, “Palmitoylation and depalmitoylation dynamics at a glance,” J. Cell Sci. 123(23), 4007–4010 (2010).
[Crossref] [PubMed]

Delahaye, J.

Deschamps, J.

Douglass, A. D.

A. D. Douglass and R. D. Vale, “Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells,” Cell 121(6), 937–950 (2005).
[Crossref] [PubMed]

Dupuis, G.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Dushek, O.

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Eckerstorfer, P.

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

Etzensperger, R.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Fort, E.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, “Full-field supercritical angle fluorescence microscopy for live cell imaging,” Opt. Lett. 36(16), 3051–3053 (2011).
[Crossref] [PubMed]

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008).
[Crossref]

Fort, S. L.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Fugger, L.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Fujiwara, T.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Gaus, K.

A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
[Crossref] [PubMed]

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Gervais, F. G.

F. G. Gervais and A. Veillette, “The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes,” Mol. Cell. Biol. 15(5), 2393–2401 (1995).
[Crossref] [PubMed]

Gray, A.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Grésillon, S.

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008).
[Crossref]

Hermens, W. T.

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Hirano, H.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Hof, M.

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Höfer, T.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Holak, T. A.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Iino, R.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Ike, H.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Jenne, D.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Kato, A.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Kessenbrock, K.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Kosugi, A.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Kusumi, A.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Lécart, S.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Lévêque-Fort, S.

Lever, M.

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

Lhotský, A.

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Ma, Y.

A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
[Crossref] [PubMed]

Maini, P. K.

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

Manstein, D. J.

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscopy,” Trends Cell Biol. 11(7), 298–303 (2001).
[Crossref] [PubMed]

Marecek, V.

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Mayet, C.

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Mets, U.

U. Mets and R. Rigler, “Submillisecond detection of single rhodamine molecules in water,” J. Fluoresc. 4(3), 259–264 (1994).
[Crossref] [PubMed]

Mund, M.

Neukirchen, D.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Nika, K.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Owen, D. M.

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Paster, W.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

Petrov, E. P.

J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[Crossref] [PubMed]

Pichler, W. J.

C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
[Crossref] [PubMed]

Polzella, P.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Reynolds, N. P.

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

Riedl, J.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Ries, J.

J. Deschamps, M. Mund, and J. Ries, “3D superresolution microscopy by supercritical angle detection,” Opt. Express 22(23), 29081–29091 (2014).
[Crossref] [PubMed]

J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[Crossref] [PubMed]

J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[Crossref] [PubMed]

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
[Crossref] [PubMed]

Rigler, R.

U. Mets and R. Rigler, “Submillisecond detection of single rhodamine molecules in water,” J. Fluoresc. 4(3), 259–264 (1994).
[Crossref] [PubMed]

Ritchie, K.

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

Rossy, J.

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Ruckstuhl, T.

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

T. Ruckstuhl, D. Verdes, C. M. Winterflood, and S. Seeger, “Simultaneous near-field and far-field fluorescence microscopy of single molecules,” Opt. Express 19(7), 6836–6844 (2011).
[Crossref] [PubMed]

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
[Crossref] [PubMed]

T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12(18), 4246–4254 (2004).
[Crossref] [PubMed]

Salek, M.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Schütz, G. J.

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

Schwille, P.

J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[Crossref] [PubMed]

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
[Crossref] [PubMed]

J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[Crossref] [PubMed]

Seeger, S.

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

T. Ruckstuhl, D. Verdes, C. M. Winterflood, and S. Seeger, “Simultaneous near-field and far-field fluorescence microscopy of single molecules,” Opt. Express 19(7), 6836–6844 (2011).
[Crossref] [PubMed]

Sixt, M.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Soldani, C.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[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]

Stockinger, H.

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[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]

Toomre, D.

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscopy,” Trends Cell Biol. 11(7), 298–303 (2001).
[Crossref] [PubMed]

Vale, R. D.

A. D. Douglass and R. D. Vale, “Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells,” Cell 121(6), 937–950 (2005).
[Crossref] [PubMed]

van der Merwe, P. A.

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

Veillette, A.

F. G. Gervais and A. Veillette, “The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes,” Mol. Cell. Biol. 15(5), 2393–2401 (1995).
[Crossref] [PubMed]

Verdes, D.

Viola, A.

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

Walker, C.

C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
[Crossref] [PubMed]

Wedlich-Soldner, R.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Weghuber, J.

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

Werb, Z.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Williamson, D. J.

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Winterflood, C. M.

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

T. Ruckstuhl, D. Verdes, C. M. Winterflood, and S. Seeger, “Simultaneous near-field and far-field fluorescence microscopy of single molecules,” Opt. Express 19(7), 6836–6844 (2011).
[Crossref] [PubMed]

Yang, Z.

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Yu, J. H.

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Zimmermann, L.

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

Biophys. J. (4)

J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
[Crossref] [PubMed]

J. Ries, E. P. Petrov, and P. Schwille, “Total Internal Reflection Fluorescence Correlation Spectroscopy: Effects of Lateral Diffusion And Surface-Generated Fluorescence,” Biophys. J. 95(1), 390–399 (2008).
[Crossref] [PubMed]

A. Benda, Y. Ma, and K. Gaus, “Self-calibrated line-scan STED-FCS to quantify lipid dynamics in model and cell membranes,” Biophys. J. 108(3), 596–609 (2015).
[Crossref] [PubMed]

J. Ries, S. Chiantia, and P. Schwille, “Accurate Determination of Membrane Dynamics with Line-Scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[Crossref] [PubMed]

Cell (1)

A. D. Douglass and R. D. Vale, “Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells,” Cell 121(6), 937–950 (2005).
[Crossref] [PubMed]

ChemPhysChem (2)

H. Ike, A. Kosugi, A. Kato, R. Iino, H. Hirano, T. Fujiwara, K. Ritchie, and A. Kusumi, “Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging,” ChemPhysChem 4(6), 620–626 (2003).
[Crossref] [PubMed]

C. M. Winterflood, T. Ruckstuhl, N. P. Reynolds, and S. Seeger, “Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection,” ChemPhysChem 13(16), 3655–3660 (2012).
[Crossref] [PubMed]

Eur. J. Immunol. (1)

C. Walker, F. Bettens, and W. J. Pichler, “T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction,” Eur. J. Immunol. 17(11), 1611–1618 (1987).
[Crossref] [PubMed]

Immunity (1)

K. Nika, C. Soldani, M. Salek, W. Paster, A. Gray, R. Etzensperger, L. Fugger, P. Polzella, V. Cerundolo, O. Dushek, T. Höfer, A. Viola, and O. Acuto, “Constitutively Active Lck kinase in T Cells Drives Antigen Receptor Signal Transduction,” Immunity 32(6), 766–777 (2010).
[Crossref] [PubMed]

J. Biol. Chem. (1)

L. Zimmermann, W. Paster, J. Weghuber, P. Eckerstorfer, H. Stockinger, and G. J. Schütz, “Direct Observation and Quantitative Analysis of Lck Exchange Between Plasma Membrane and Cytosol in Living T Cells,” J. Biol. Chem. 285(9), 6063–6070 (2010).
[Crossref] [PubMed]

J. Cell Sci. (1)

E. Conibear and N. G. Davis, “Palmitoylation and depalmitoylation dynamics at a glance,” J. Cell Sci. 123(23), 4007–4010 (2010).
[Crossref] [PubMed]

J. Fluoresc. (1)

U. Mets and R. Rigler, “Submillisecond detection of single rhodamine molecules in water,” J. Fluoresc. 4(3), 259–264 (1994).
[Crossref] [PubMed]

J. Phys. D Appl. Phys. (1)

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D Appl. Phys. 41(1), 013001 (2008).
[Crossref]

Langmuir (1)

A. Benda, M. Beneš, V. Mareček, A. Lhotský, W. T. Hermens, and M. Hof, “How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy,” Langmuir 19(10), 4120–4126 (2003).
[Crossref]

Mol. Cell. Biol. (1)

F. G. Gervais and A. Veillette, “The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes,” Mol. Cell. Biol. 15(5), 2393–2401 (1995).
[Crossref] [PubMed]

Nat. Immunol. (1)

J. Rossy, D. M. Owen, D. J. Williamson, Z. Yang, and K. Gaus, “Conformational states of the kinase Lck regulate clustering in early T cell signaling,” Nat. Immunol. 14(1), 82–89 (2012).
[Crossref] [PubMed]

Nat. Methods (1)

J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb, M. Sixt, and R. Wedlich-Soldner, “Lifeact: a versatile marker to visualize F-actin,” Nat. Methods 5(7), 605–607 (2008).
[Crossref] [PubMed]

Nat. Photonics (1)

N. Bourg, C. Mayet, G. Dupuis, T. Barroca, P. Bon, S. Lécart, E. Fort, and S. L. Fort, “Direct optical nanoscopy with axially localized detection,” Nat. Photonics 9(9), 587–593 (2015).
[Crossref]

Nat. Protoc. (1)

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

Nat. Rev. Immunol. (1)

M. Lever, P. K. Maini, P. A. van der Merwe, and O. Dushek, “Phenotypic models of T cell activation,” Nat. Rev. Immunol. 14(9), 619–629 (2014).
[Crossref] [PubMed]

Opt. Express (3)

Opt. Lett. (1)

Trends Cell Biol. (1)

D. Toomre and D. J. Manstein, “Lighting up the cell surface with evanescent wave microscopy,” Trends Cell Biol. 11(7), 298–303 (2001).
[Crossref] [PubMed]

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Figures (5)

Fig. 1
Fig. 1 Schematic setup of the SAF microscope. (a) The light path of the SAF objective. A collimated excitation laser (blue) is focused to a diffraction-limited point at by a central aspheric lens with f = 3 mm. Emission (green) at low angles (UAF) is collected by this same lens with higher-angle emission (SAF) collected by a confocal parabolic mirror. Intermediate angles of emission are blocked by an opaque ring mask in the back focal plane of the objective. (b) The collimated SAF and UAF emission beams are split by a mirror placed at the conjugate sample plane of the objective. In the mirror, a 3 mm x 4.5 mm elliptical hole was made to pass UAF and reflect SAF. Finally, the separated UAF and SAF fluorescence are each refocused onto the active area of the photodetectors.
Fig. 2
Fig. 2 SAF measurements in lipid bilayers. Representative UAF (a) and SAF (b) images of live COS-7 cells expressing Lifeact-Venus. The images are colored blue to red to indicate low to high pixel intensity. Scale bar = 5 μm. Data in a and b are representative of 5 cells. (c, d) x-z images of COS-7 cells expressing Lck10-EGFP recorded in the UAF (c) and SAF (d) channels. Scale bar = 3 μm (e) Quantification of diffusion time of DHPE-OG488 in a supported lipid bilayer (black, left axis) and SAF FCS beam waist (blue, right axis) as measured by z-scan SAF FCS. Measurements were taken at different focal positions 0.5 µm apart. Data (symbols) were fitted to a parabolic function (lines, Eqs. (6) and (7)) [20]. R2 = 0.962 for diffusion time and R2 = 0.970 for particle number, respectively) (f) SAF FCS, UAF FCS and cross-correlation of SAF and UAF of DHPE-OG488 (1 nM) in a supported lipid bilayer. (g) SAF FCS, UAF FCS and cross-correlation of SAF and UAF of ATTO 488 dye (100 nM) in solution above a supported lipid bilayer containing DHPE-OG488 (1 nM). (h) SAF FCS, UAF FCS and cross-correlation of SAF and UAF of ATTO 488 (100 nM) in solution. Data in f, g, and h is representative of 6 measurements.
Fig. 3
Fig. 3 Comparison of LS FCS, SAF FCS and UAF FCS measurements of model and cell membranes. (a) Autocorrelation curves obtained by LS FCS (green), SAF FCS (black) and UAF FCS (blue) of DHPE-OG488 (20 nM) in supported lipid bilayers. The data was fitted to a one-species two-dimensional free diffusion model (red lines). (b, c) Diffusion coefficients (b) and concentrations (c) obtained from LS FCS, SAF FCS and UAF FCS measurements of DHPE-OG488 in lipid bilayers. Each symbol represents an independent experiment (n = 6), horizontal and vertical bars represent means and standard errors (*P<0.05, two-tailed t-test). (d) Fitting of the LS FCS auto-correlation curve (black line) of Lck10-EGFP in COS-7 cells to one-species (red line) and two-species (blue line) two-dimensional diffusion models. (e, g) Diffusion coefficients (e), and ratio of amplitudes (g) of the slow and fast diffusion components (P = 0.35 and 0.31 for the slow and fast Lck10-EGFP components, respectively) of Lck10-EGFP obtained by LS FCS and SAF FCS. Values were extracted by fitting autocorrelation curves to two-species diffusion model, yielding diffusion coefficients for slow (triangle and stars) and fast (circle and diamond) diffusion components for both LS FCS and SAF FCS measurements. Each symbol represents an independent experiment (n = 11), horizontal and vertical bars represent means and standard errors (*P = 0.012 in g). (f) SAF FCS, UAF FCS and cross-correlation of SAF and UAF of free diffusing cytosolic EGFP in COS-7 cells. Data shown in f is representative of 6 measurements.
Fig. 4
Fig. 4 Analysis of auto-correlation curves by the fitting-free T1/2 method. (a) Reprehensive example of analysis of Lck10-EGFP diffusion time by diffusion model fitting or T1/2 method, see Methods. This T1/2 value is indicated by dotted lines in the graph. The amplitude of the correlation curve is defined as the G(γ) value at 200 µs. (b, c) Plot of diffusion coefficient (b) and concentration (c) of Lck10 EGFP in COS-7 cells by SAF and LS FCS by the T1/2 method. The mean (black solid line) and standard error bar are indicated (n = 10, *P = 0.039 and 0.60 in b and c, respectively).
Fig. 5
Fig. 5 Diffusion analysis of Lck in resting and activated T cells with SAF FCS. (a) Auto-correlation curve (black line) of Lck-EGFP in activated T cells fitted to a one-species two-dimensional diffiusion model (blue line) and two-species two-dimensional (red line) diffusion model. The fitting residuals are plotted below. (b, c) Diffusion coefficients (b) and the ratio of amplitudes of the slow to fast components (c) of Lck-EGFP in resting and activated Jurkat cells obtained by fitting SAF FCS data to two-species diffusion model, resulting in values for slow (triangle and stars) and fast (circle and diamond) diffusing Lck-EGFP species. Each symbol represents an independent experiment (n = 15), horizontal and vertical bars represent means and standard errors (P = 0.56 and 0.25 for the slow and fast Lck-EGFP components, respectively in b, *P = 0.030 in c, two-tailed t-test). (d, e) Average diffusion coefficient (d) and average concentrations (e) of Lck-GFP extracted by the T1/2 method of the data sets shown in (b). ns, not significant; *P = 0.04 in (d) and P = 0.62 in (e). (f, g) Average diffusion coefficient (f) and average concentrations (g) of Lck-EGFP extracted by the T1/2 method for the signal collected in the UAF channel. Each symbol represents an independent experiment (n = 11), horizontal and vertical bars means and standard errors (P = 0.07 in f and *P = 0.015 in g).

Equations (7)

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G(τ)=1+ δI(t)δI(t+τ) I (t) 2
G (τ) cross =1+ δ I SAF (t)δ I UAF (t+τ) I SAF (t) I UAF (t)
G(τ)= 1 Cπ ω 0 2 ( 1+ 4 τ D ω 0 2 ) 1
D= ω 0 2 4 τ D
G= A 1 2 G 1 + A 2 2 G 2 ( A 1 + A 2 )
τ D = ω 0 2 4D ( 1+ λ 2 Δ z 2 π 2 n 2 ω 0 2 )
PN=πc ω 0 2 ( 1+ λ 2 Δ z 2 π 2 n 2 ω 0 4 )

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