Abstract

Total-internal-reflection fluorescence (TIRF) microscopy provides high optical-sectioning capability and a good signal-contrast ratio for structures near the surfaces of cells. In recent years, several improvements have been developed, such as variable-angle TIRF (VA-TIRF) and spinning TIRF (sp-TIRF), which permit quantitative image analysis and address non-uniform scattering fringes, respectively. Here, we present a dual-color DMD-based shadowless-illuminated variable-angle TIRF (siva-TIRF) system that provides a uniform illumination field. By adjusting the incidence angle of the illuminating laser on the back focal plane (BFP) of the objective, we can rapidly illuminate biological samples in layers of various thicknesses in TIRF or hollow-cone epi-fluorescence mode. Compared with other methods of accomplishing VA-TIRF/sp-TIRF illumination, our system is simple to build and cost-effective, and it provides optimal multi-plane dual-color images. By showing spatiotemporal correlated movement of clathrin-coated structures with microtubule filaments from various layers of live cells, we demonstrate that cortical microtubules are important spatial regulators of clathrin-coated structures. Moreover, our system can be used to prove superb axial information of three-dimensional movement of structures near the plasma membrane within live cells.

© 2014 Optical Society of America

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  1. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol.89(1), 141–145 (1981).
    [CrossRef] [PubMed]
  2. C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell121(4), 593–606 (2005).
    [CrossRef] [PubMed]
  3. J. A. Steyer, H. Horstmann, and W. Almers, “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature388(6641), 474–478 (1997).
    [CrossRef] [PubMed]
  4. R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
    [CrossRef] [PubMed]
  5. A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
    [CrossRef] [PubMed]
  6. J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
    [CrossRef] [PubMed]
  7. M. van ’t Hoff, V. de Sars, and M. Oheim, “A programmable light engine for quantitative single molecule TIRF and HILO imaging,” Opt. Express16(22), 18495–18504 (2008).
    [CrossRef] [PubMed]
  8. D. Axelrod and G. M. Omann, “Combinatorial microscopy,” Nat. Rev. Mol. Cell Biol.7(12), 944–952 (2006).
    [CrossRef] [PubMed]
  9. A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci.123(21), 3621–3628 (2010).
    [CrossRef] [PubMed]
  10. D. Zenisek, J. A. Steyer, and W. Almers, “Transport, capture and exocytosis of single synaptic vesicles at active zones,” Nature406(6798), 849–854 (2000).
    [CrossRef] [PubMed]
  11. J. Lin and A. D. Hoppe, “Uniform total internal reflection fluorescence illumination enables live cell fluorescence resonance energy transfer microscopy,” Microsc. Microanal.19(2), 350–359 (2013).
    [CrossRef] [PubMed]
  12. F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
    [CrossRef] [PubMed]
  13. M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods5(2), 159–161 (2008).
    [CrossRef] [PubMed]
  14. P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
    [CrossRef] [PubMed]
  15. G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
    [CrossRef] [PubMed]
  16. D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
    [CrossRef] [PubMed]
  17. R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett.33(14), 1629–1631 (2008).
    [CrossRef] [PubMed]
  18. D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
    [CrossRef] [PubMed]
  19. A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
    [CrossRef] [PubMed]
  20. A. Rohrbach, “Observing secretory granules with a multiangle evanescent wave microscope,” Biophys. J.78(5), 2641–2654 (2000).
    [CrossRef] [PubMed]
  21. D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
    [CrossRef] [PubMed]
  22. B. P. Olveczky, N. Periasamy, and A. S. Verkman, “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy,” Biophys. J.73(5), 2836–2847 (1997).
    [CrossRef] [PubMed]

2014

D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
[CrossRef] [PubMed]

2013

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

J. Lin and A. D. Hoppe, “Uniform total internal reflection fluorescence illumination enables live cell fluorescence resonance energy transfer microscopy,” Microsc. Microanal.19(2), 350–359 (2013).
[CrossRef] [PubMed]

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

2012

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

2010

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci.123(21), 3621–3628 (2010).
[CrossRef] [PubMed]

2008

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods5(2), 159–161 (2008).
[CrossRef] [PubMed]

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
[CrossRef] [PubMed]

R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett.33(14), 1629–1631 (2008).
[CrossRef] [PubMed]

M. van ’t Hoff, V. de Sars, and M. Oheim, “A programmable light engine for quantitative single molecule TIRF and HILO imaging,” Opt. Express16(22), 18495–18504 (2008).
[CrossRef] [PubMed]

2006

A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
[CrossRef] [PubMed]

D. Axelrod and G. M. Omann, “Combinatorial microscopy,” Nat. Rev. Mol. Cell Biol.7(12), 944–952 (2006).
[CrossRef] [PubMed]

2005

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell121(4), 593–606 (2005).
[CrossRef] [PubMed]

2003

P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
[CrossRef] [PubMed]

2002

D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
[CrossRef] [PubMed]

2000

D. Zenisek, J. A. Steyer, and W. Almers, “Transport, capture and exocytosis of single synaptic vesicles at active zones,” Nature406(6798), 849–854 (2000).
[CrossRef] [PubMed]

A. Rohrbach, “Observing secretory granules with a multiangle evanescent wave microscope,” Biophys. J.78(5), 2641–2654 (2000).
[CrossRef] [PubMed]

1997

J. A. Steyer, H. Horstmann, and W. Almers, “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature388(6641), 474–478 (1997).
[CrossRef] [PubMed]

B. P. Olveczky, N. Periasamy, and A. S. Verkman, “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy,” Biophys. J.73(5), 2836–2847 (1997).
[CrossRef] [PubMed]

1996

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

1985

F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
[CrossRef] [PubMed]

1981

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol.89(1), 141–145 (1981).
[CrossRef] [PubMed]

Almers, W.

D. Zenisek, J. A. Steyer, and W. Almers, “Transport, capture and exocytosis of single synaptic vesicles at active zones,” Nature406(6798), 849–854 (2000).
[CrossRef] [PubMed]

J. A. Steyer, H. Horstmann, and W. Almers, “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature388(6641), 474–478 (1997).
[CrossRef] [PubMed]

Axelrod, D.

D. Axelrod and G. M. Omann, “Combinatorial microscopy,” Nat. Rev. Mol. Cell Biol.7(12), 944–952 (2006).
[CrossRef] [PubMed]

A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
[CrossRef] [PubMed]

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol.89(1), 141–145 (1981).
[CrossRef] [PubMed]

Beck, M.

Belyaev, Y.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
[CrossRef] [PubMed]

Benmerah, A.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Beuthan, J.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

Castro-Castro, A.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Chavrier, P.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Chitnis, A. B.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Combs, C. A.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Dalle Nogare, D.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Dan, D.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

de Sars, V.

Ewers, H.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
[CrossRef] [PubMed]

Fiolka, R.

R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
[CrossRef] [PubMed]

R. Fiolka, M. Beck, and A. Stemmer, “Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator,” Opt. Lett.33(14), 1629–1631 (2008).
[CrossRef] [PubMed]

Fischer, R. S.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Franco, M.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Gao, P.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Helfmann, J.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

Herrig, M.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

Hoppe, A. D.

J. Lin and A. D. Hoppe, “Uniform total internal reflection fluorescence illumination enables live cell fluorescence resonance energy transfer microscopy,” Microsc. Microanal.19(2), 350–359 (2013).
[CrossRef] [PubMed]

Horstmann, H.

J. A. Steyer, H. Horstmann, and W. Almers, “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature388(6641), 474–478 (1997).
[CrossRef] [PubMed]

Imamoto, N.

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods5(2), 159–161 (2008).
[CrossRef] [PubMed]

Irondelle, M.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Johnson, D. S.

D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
[CrossRef] [PubMed]

Keyel, P. A.

P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
[CrossRef] [PubMed]

Lanni, F.

F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
[CrossRef] [PubMed]

Lei, M.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Lin, J.

J. Lin and A. D. Hoppe, “Uniform total internal reflection fluorescence illumination enables live cell fluorescence resonance energy transfer microscopy,” Microsc. Microanal.19(2), 350–359 (2013).
[CrossRef] [PubMed]

Loerke, D.

D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
[CrossRef] [PubMed]

Mattheyses, A. L.

D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
[CrossRef] [PubMed]

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci.123(21), 3621–3628 (2010).
[CrossRef] [PubMed]

A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
[CrossRef] [PubMed]

Meas-Yedid, V.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[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,” Cell121(4), 593–606 (2005).
[CrossRef] [PubMed]

Minet, O.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

Mione, M.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Montagnac, G.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Müller, G.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, “The spatial variation of the refractive index in biological cells,” Phys. Med. Biol.41(3), 369–382 (1996).
[CrossRef] [PubMed]

Nachury, M. V.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Oheim, M.

M. van ’t Hoff, V. de Sars, and M. Oheim, “A programmable light engine for quantitative single molecule TIRF and HILO imaging,” Opt. Express16(22), 18495–18504 (2008).
[CrossRef] [PubMed]

D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
[CrossRef] [PubMed]

Olivo-Marin, J. C.

G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

Olveczky, B. P.

B. P. Olveczky, N. Periasamy, and A. S. Verkman, “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy,” Biophys. J.73(5), 2836–2847 (1997).
[CrossRef] [PubMed]

Omann, G. M.

D. Axelrod and G. M. Omann, “Combinatorial microscopy,” Nat. Rev. Mol. Cell Biol.7(12), 944–952 (2006).
[CrossRef] [PubMed]

Parekh, S. H.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

Periasamy, N.

B. P. Olveczky, N. Periasamy, and A. S. Verkman, “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy,” Biophys. J.73(5), 2836–2847 (1997).
[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,” Cell121(4), 593–606 (2005).
[CrossRef] [PubMed]

Qi, Y.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Rappoport, J. Z.

A. L. Mattheyses, S. M. Simon, and J. Z. Rappoport, “Imaging with total internal reflection fluorescence microscopy for the cell biologist,” J. Cell Sci.123(21), 3621–3628 (2010).
[CrossRef] [PubMed]

Rohrbach, A.

A. Rohrbach, “Observing secretory granules with a multiangle evanescent wave microscope,” Biophys. J.78(5), 2641–2654 (2000).
[CrossRef] [PubMed]

Sakata-Sogawa, K.

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A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
[CrossRef] [PubMed]

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G. Montagnac, V. Meas-Yedid, M. Irondelle, A. Castro-Castro, M. Franco, T. Shida, M. V. Nachury, A. Benmerah, J. C. Olivo-Marin, and P. Chavrier, “αTAT1 catalyses microtubule acetylation at clathrin-coated pits,” Nature502(7472), 567–570 (2013).
[CrossRef] [PubMed]

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A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

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D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
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D. Zenisek, J. A. Steyer, and W. Almers, “Transport, capture and exocytosis of single synaptic vesicles at active zones,” Nature406(6798), 849–854 (2000).
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D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
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F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
[CrossRef] [PubMed]

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A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

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M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods5(2), 159–161 (2008).
[CrossRef] [PubMed]

Toledo-Crow, R.

D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
[CrossRef] [PubMed]

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P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
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B. P. Olveczky, N. Periasamy, and A. S. Verkman, “Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy,” Biophys. J.73(5), 2836–2847 (1997).
[CrossRef] [PubMed]

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F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
[CrossRef] [PubMed]

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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

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P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
[CrossRef] [PubMed]

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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

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D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Yan, S.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Yang, Y.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Yao, B.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Ye, T.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

York, A. G.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
[CrossRef] [PubMed]

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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,” Cell121(4), 593–606 (2005).
[CrossRef] [PubMed]

D. Zenisek, J. A. Steyer, and W. Almers, “Transport, capture and exocytosis of single synaptic vesicles at active zones,” Nature406(6798), 849–854 (2000).
[CrossRef] [PubMed]

Zhao, W.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Zumbusch, A.

D. Dan, M. Lei, B. Yao, W. Wang, M. Winterhalder, A. Zumbusch, Y. Qi, L. Xia, S. Yan, Y. Yang, P. Gao, T. Ye, and W. Zhao, “DMD-based LED-illumination super-resolution and optical sectioning microscopy,” Sci. Rep.3, 1116 (2013).
[CrossRef] [PubMed]

Biophys. J.

D. S. Johnson, R. Toledo-Crow, A. L. Mattheyses, and S. M. Simon, “Polarization-Controlled TIRFM with Focal Drift and Spatial Field Intensity Correction,” Biophys. J.106(5), 1008–1019 (2014).
[CrossRef] [PubMed]

A. Rohrbach, “Observing secretory granules with a multiangle evanescent wave microscope,” Biophys. J.78(5), 2641–2654 (2000).
[CrossRef] [PubMed]

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[CrossRef] [PubMed]

Cell

C. J. Merrifield, D. Perrais, and D. Zenisek, “Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells,” Cell121(4), 593–606 (2005).
[CrossRef] [PubMed]

J. Biol. Chem.

P. A. Keyel, S. C. Watkins, and L. M. Traub, “Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy,” J. Biol. Chem.279(13), 13190–13204 (2003).
[CrossRef] [PubMed]

J. Cell Biol.

F. Lanni, A. S. Waggoner, and D. L. Taylor, “Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy,” J. Cell Biol.100(4), 1091–1102 (1985).
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[CrossRef] [PubMed]

J. Neurosci. Methods

D. Loerke, W. Stühmer, and M. Oheim, “Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation,” J. Neurosci. Methods119(1), 65–73 (2002).
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J. Lin and A. D. Hoppe, “Uniform total internal reflection fluorescence illumination enables live cell fluorescence resonance energy transfer microscopy,” Microsc. Microanal.19(2), 350–359 (2013).
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R. Fiolka, Y. Belyaev, H. Ewers, and A. Stemmer, “Even illumination in total internal reflection fluorescence microscopy using laser light,” Microsc. Res. Tech.71(1), 45–50 (2008).
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A. L. Mattheyses, K. Shaw, and D. Axelrod, “Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle,” Microsc. Res. Tech.69(8), 642–647 (2006).
[CrossRef] [PubMed]

Nat. Methods

M. Tokunaga, N. Imamoto, and K. Sakata-Sogawa, “Highly inclined thin illumination enables clear single-molecule imaging in cells,” Nat. Methods5(2), 159–161 (2008).
[CrossRef] [PubMed]

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods9(7), 749–754 (2012).
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Nature

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[CrossRef] [PubMed]

J. A. Steyer, H. Horstmann, and W. Almers, “Transport, docking and exocytosis of single secretory granules in live chromaffin cells,” Nature388(6641), 474–478 (1997).
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[CrossRef] [PubMed]

Supplementary Material (1)

» Media 1: AVI (118042 KB)     

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Figures (5)

Fig. 1
Fig. 1

Schematic of the siva-TIRF microscope. AOM: acousto-optical modulator, CL: collimating lens, QWP: quarter-wave plate, TD: tunable diaphragm, RM: reflection mirror, SM: stop mask, OL: objective lens, DM1 and DM2: dichroic mirrors, EF: emission filter, TL: tube lens, SP: split diaphragm. Insert (a) shows the continuously alternating incident laser beams at different positions of the BFP during one camera-exposure cycle, which created uniform illumination in the TIRF and HOCE modes. Insert (b) shows the positions of the focal spots of the incident laser on the BFP. Insert (c) describes the working principle of the operation of a DMD as a grid grating. Insert (d) describes the schematic of the generation of a fine non-integral period of the DMD pattern. ρ is the physical size of one grating period (14 μm is the pixel size of the DMD).The integral ωb consisted of parallel lines of “on” and “off” pixels; the non-integral ω was generated by rotating the basic ωb patterns through various angles δ.

Fig. 2
Fig. 2

Adjustment of the incidence angle of the excitation light and generation of even illumination using the DMD. (a) Measurement of the correlation between the incidence angle θ and the pattern period ω of the DMD. Upper left: the incidence angle of the excitation light was measured from its path on a glass cube. Lower left: measured correlation between the incidence angle θ and ω. Data were collected for ω from 4.0 lines to 5.0 lines for 488 nm and from 4.5 to 5.5 for 561 nm, which were equivalent to the penetration thickness from 70 nm to 10 μm for both 488 nm and 561 nm. The θ value was the average of the six laser beams created using the same ω but different azimuthal angles, and ∆θ was the largest deviation among the six measurements. Upper middle and right: HOCE and TIRF illuminations. Three lower right plots: calculation of the penetration depths dT in TIRF, dH in HOCE and in the critical phase as a function of θ. (b) Measurement of the laser intensity. Left: diagram of the six laser focal spots on the BFP separated by equal divisions. Right: there were deviations in laser energy of approximately ± 3.5% among the focal spots at the BFP produced using the same ω for the 488 nm laser and corresponding deviations of ± 5% for the 561 nm laser. Different values of ω (and therefore different incidence angles θ) also did not significantly affect the laser intensity at the focal spots. (c) The illumination field became homogeneous when the samples were excited by incident light originating from six focal spots on the BFP. One beam, two beams and six beams of 488 nm and 561 nm light were used to excite the Na-FITC and Rose Bengal samples, respectively. The scale bar in (c) represents 10 μm; the size of the images is 400 × 400 pixels.

Fig. 3
Fig. 3

Growth of microtubules from 150 nm to less than 80 nm from the membrane in live INS-1 cell (Media 1). (a)−(c) Fluorescence images of microtubules labeled with EB3-GFP under TIRF illumination with penetration depths of 80 nm (a), 100 nm (b) and 150 nm (c). (d) Color-merged image of (a)−(c). (a)−(d) are also shown in Media 1. (e) Time-lapse montage of the enlarged region indicated by the yellow box in (d); the arrows show the plus end of a microtubule approaching from deeper than 150 nm to the below 80 nm TIRF zone and then retreating once again. The scale bars in (a)−(d) represent 10 μm, whereas that in (e) represents 2 μm. Each image in the sequence is separated by 2 s from the preceding image.

Fig. 4
Fig. 4

Rapid motion of clathrin in a live cell from 500 nm to less than 80 nm from the membrane. (a) and (b) The spatial distribution of clathrin in an INS-1 cell under 80 nm (a), and 500 nm (b) TIRF illumination. (c) Merged images of clathrin puncta observed at different penetration depths. (d) Rapid motion of a clathrin punctum from deep in the cell to the plasma membrane. This montage of time-lapse images corresponds to the region indicated by a yellow frame in (c) with a time interval of 200 ms. The blue arrow represents the moving clathrin. The scale bars in (a)–(c) represents 10 μm, whereas that in (c) represents 500 nm

Fig. 5
Fig. 5

Distribution of microtubules and clathrin from 100 nm to 500 nm away from the membrane in a live cell, represented by dual-color images. (a)−(c) EB3 in an INS-1 cell under TIRF illumination with penetration depths of 80 nm (a), 150 nm (b), and 500 nm (c). (d)−(f) Clathrin in the same INS-1 cell with penetration depths of 80 nm (d), 150 nm (e), and 500 nm (f). (g) Time-lapse montage of the enlarged region indicated by the yellow box in (a) and (d). The arrows show a CCP that co-localized with a microtubule filament and remained stationary during the time of recording. (h) Time-lapse montage of the enlarged region indicated by the blue box in (c) and (f). The arrows show a CCV that co-localized with a microtubule filament and moved along the filament during the time of recording. Scale bar in (a)−(f) represents 10 μm, whereas that in (g)−(h) represent 1 μm. Each image in the sequence in (g) and (h) is separated by 6 s from the preceding image. Red: clathrin, Green: EB3.

Equations (3)

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I(z)=I(0)exp(z/d)
d T = λ 4π ( n 1 2 sin 2 θ n 2 2 ) 1/2
dH= D0 2Mtan [ sin 1 (sinθ n 2 / n 1 )] 1/2

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