Abstract

Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within 100nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the- objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the 50nm deep evanescent field.

© 2009 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  3. N. Bobroff, “Position measurement with a resolution and noise-limited instrument,” Rev. Sci. Instrum. 57, 1152-1157(1986).
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  4. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5 nm localization,” Science 300, 2061-2065 (2003).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  7. J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  14. D. Axelrod and G. M. Omann, “Combinatorial microscopy,” Nat. Rev. Mol. Cell Biol. 7, 944-952 (2006).
    [CrossRef] [PubMed]
  15. A. L. Stout and D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28, 5237-5242 (1989).
    [CrossRef] [PubMed]
  16. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89, 141-145(1981).
    [CrossRef] [PubMed]
  17. T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  20. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. A 253, 358-379 (1959).
    [CrossRef]
  21. T. P. Burghardt and N. L. Thompson, “Evanescent intensity of a focused Gaussian light beam undergoing total internal reflection in a prism,” Opt. Eng. 23, 62-67 (1984).
  22. E. H. Hellen and D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4, 337-350(1987).
    [CrossRef]
  23. D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557-573 (1979).
    [CrossRef] [PubMed]
  24. T. P. Burghardt and D. Axelrod, “Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics,” Biophys. J. 33, 455-467 (1981).
    [CrossRef] [PubMed]
  25. A. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging,” J. Biomed. Opt. 10, 054007 (2005).
    [CrossRef] [PubMed]
  26. D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
    [CrossRef] [PubMed]

2009 (1)

T. P. Burghardt and K. Ajtai, “Mapping microscope object polarized emission to the back focal plane pattern,” J. Biomed. Opt. 14, 034036 (2009).
[CrossRef] [PubMed]

2007 (1)

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

2006 (5)

T. P. Burghardt, K. Ajtai, and J. Borejdo, “In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy,” Biochemistry 45, 4058-4068 (2006).
[CrossRef] [PubMed]

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

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

2005 (3)

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nature Methods 2, 905-909(2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. USA 102, 13081-13086 (2005).
[CrossRef] [PubMed]

A. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging,” J. Biomed. Opt. 10, 054007 (2005).
[CrossRef] [PubMed]

2004 (3)

2003 (2)

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

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Meth. Enzymol. 361, 1-33 (2003).
[CrossRef] [PubMed]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

1989 (1)

1987 (1)

1986 (1)

N. Bobroff, “Position measurement with a resolution and noise-limited instrument,” Rev. Sci. Instrum. 57, 1152-1157(1986).
[CrossRef]

1984 (2)

T. P. Burghardt and N. L. Thompson, “Evanescent intensity of a focused Gaussian light beam undergoing total internal reflection in a prism,” Opt. Eng. 23, 62-67 (1984).

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
[CrossRef] [PubMed]

1981 (2)

T. P. Burghardt and D. Axelrod, “Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics,” Biophys. J. 33, 455-467 (1981).
[CrossRef] [PubMed]

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

1979 (1)

D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557-573 (1979).
[CrossRef] [PubMed]

1974 (1)

A. Yoshida and T. Asakura, “Electromagnetic field near the focus of gaussian beams,” Optik (Jena) 41, 281-292 (1974).

1959 (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. A 253, 358-379 (1959).
[CrossRef]

Ajtai, K.

T. P. Burghardt and K. Ajtai, “Mapping microscope object polarized emission to the back focal plane pattern,” J. Biomed. Opt. 14, 034036 (2009).
[CrossRef] [PubMed]

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

T. P. Burghardt, K. Ajtai, and J. Borejdo, “In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy,” Biochemistry 45, 4058-4068 (2006).
[CrossRef] [PubMed]

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Asakura, T.

A. Yoshida and T. Asakura, “Electromagnetic field near the focus of gaussian beams,” Optik (Jena) 41, 281-292 (1974).

Axelrod, D.

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

A. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging,” J. Biomed. Opt. 10, 054007 (2005).
[CrossRef] [PubMed]

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Meth. Enzymol. 361, 1-33 (2003).
[CrossRef] [PubMed]

A. L. Stout and D. Axelrod, “Evanescent field excitation of fluorescence by epi-illumination microscopy,” Appl. Opt. 28, 5237-5242 (1989).
[CrossRef] [PubMed]

E. H. Hellen and D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4, 337-350(1987).
[CrossRef]

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
[CrossRef] [PubMed]

T. P. Burghardt and D. Axelrod, “Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics,” Biophys. J. 33, 455-467 (1981).
[CrossRef] [PubMed]

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

D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557-573 (1979).
[CrossRef] [PubMed]

Bobroff, N.

N. Bobroff, “Position measurement with a resolution and noise-limited instrument,” Rev. Sci. Instrum. 57, 1152-1157(1986).
[CrossRef]

Borejdo, J.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

T. P. Burghardt, K. Ajtai, and J. Borejdo, “In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy,” Biochemistry 45, 4058-4068 (2006).
[CrossRef] [PubMed]

Burghardt, T. P.

T. P. Burghardt and K. Ajtai, “Mapping microscope object polarized emission to the back focal plane pattern,” J. Biomed. Opt. 14, 034036 (2009).
[CrossRef] [PubMed]

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

T. P. Burghardt, K. Ajtai, and J. Borejdo, “In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy,” Biochemistry 45, 4058-4068 (2006).
[CrossRef] [PubMed]

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

T. P. Burghardt and N. L. Thompson, “Evanescent intensity of a focused Gaussian light beam undergoing total internal reflection in a prism,” Opt. Eng. 23, 62-67 (1984).

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
[CrossRef] [PubMed]

T. P. Burghardt and D. Axelrod, “Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics,” Biophys. J. 33, 455-467 (1981).
[CrossRef] [PubMed]

Calander, N.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Chan, D. K.

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

Charlesworth, J. E.

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Eggeling, C.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Forkey, J. N.

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

Goldman, Y. E.

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

Gryczynski, I.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Gryczynski, Z.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. USA 102, 13081-13086 (2005).
[CrossRef] [PubMed]

Ha, T.

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

Halstead, M. F.

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

Hell, S. W.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Hellen, E. H.

Jan, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Keller, J.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

Li, J.

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

Lieb, M. A.

Luhrmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Mattheyses, A.

A. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces investigated with back focal plane imaging,” J. Biomed. Opt. 10, 054007 (2005).
[CrossRef] [PubMed]

McKinney, S. A.

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

Medda, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Muthu, P.

J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu, and I. Gryczynski, “Application of surface plasmon coupled emission to study of muscle,” Biophys. J. 91, 2626-2635 (2006).
[CrossRef] [PubMed]

Novotny, L.

Omann, G. M.

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

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. A 253, 358-379 (1959).
[CrossRef]

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Luhrmann, R. Jan, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. USA 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Ruckstuhl, T.

Seeger, S.

Selvin, P. R.

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

Shaner, N. C.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nature Methods 2, 905-909(2005).
[CrossRef] [PubMed]

Steinbach, P. A.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nature Methods 2, 905-909(2005).
[CrossRef] [PubMed]

Stout, A. L.

Tarara, J. E.

T. P. Burghardt, J. E. Charlesworth, M. F. Halstead, J. E. Tarara, and K. Ajtai, “In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy,” Biophys. J. 90, 4662-4671 (2006).
[CrossRef] [PubMed]

Thompson, N. L.

T. P. Burghardt and N. L. Thompson, “Evanescent intensity of a focused Gaussian light beam undergoing total internal reflection in a prism,” Opt. Eng. 23, 62-67 (1984).

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
[CrossRef] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

Tsien, R. Y.

N. C. Shaner, P. A. Steinbach, and R. Y. Tsien, “A guide to choosing fluorescent proteins,” Nature Methods 2, 905-909(2005).
[CrossRef] [PubMed]

Verdes, D.

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. A 253, 358-379 (1959).
[CrossRef]

Yildiz, A.

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

Yoshida, A.

A. Yoshida and T. Asakura, “Electromagnetic field near the focus of gaussian beams,” Optik (Jena) 41, 281-292 (1974).

Zavislan, J. M.

Zheng, Y.

T. P. Burghardt, K. Ajtai, D. K. Chan, M. F. Halstead, J. Li, and Y. Zheng, “GFP tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber,” Biophys. J. 93, 2226-2239 (2007).
[CrossRef] [PubMed]

Annu Rev Biophys Bioeng (1)

D. Axelrod, T. P. Burghardt, and N. L. Thompson, “Total internal reflection fluorescence,” Annu Rev Biophys Bioeng 13, 247-268 (1984).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biochemistry (1)

T. P. Burghardt, K. Ajtai, and J. Borejdo, “In situ single molecule imaging with attoliter detection using objective total internal reflection confocal microscopy,” Biochemistry 45, 4058-4068 (2006).
[CrossRef] [PubMed]

Biophys. J. (6)

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Figures (10)

Fig. 1
Fig. 1

Schematic aoTIRF diagrams for (a) upright and (b) inverted microscope configurations. Dashed lines show the beam path through the prisms to the glass/aqueous interface where the exciting beam totally internally reflects. The inverted configuration has two other total internal reflections at glass/air interfaces. Coverslips and Littrow prisms are optically coupled by immersion oil. The upright microscope configuration also shows the beam conditioning elements consisting of a beam waist reducer from 10 × and 20 × microscope objectives and a low-aperture focusing lens. Beam conditioning is needed in both upright and inverted microscope configurations but is shown only for the upright.

Fig. 2
Fig. 2

Detailed schematic aoTIRF upright microscope configuration. Symbols defining distances or angles are used in the text. Distance C is 0.15 mm for a #1 coverslip. The approach-limiting edge requires θ 2 3 deg and is the strictest constraint on laser beam shape. The upright configuration works with both small and large base Littrow prisms. Part of the objective is shown with the incident laser beam path. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.

Fig. 3
Fig. 3

Detailed schematic aoTIRF inverted microscope configuration. Symbols defining distances or angles used in Fig. 2 are equivalently defined here. Numeric distances are given in millimeters. Distance B is 0.21 mm for a #2 coverslip. Oil is confined by the objective, the lower edge of the #1 coverslip, the short edges of the #2 coverslips, and the oil retainers. The inverted configuration requires the large base Littrow prisms. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.

Fig. 4
Fig. 4

Two views of the exciting laser beam intensity profile (rippled surface) and (FOV plane for the microscope objective near the focal plane for the exciting light. The x p and y p axes lie in the focal plane of the exciting light with z p in the direction of light propagation and y p in the incidence plane at the glass/aqueous interface. The vertical axis is intensity in arbitrary units for the intensity profile and micrometers for the (a)  y p or (b)  x p spatial dimensions.

Fig. 5
Fig. 5

The exciting laser beam cross section for various z p values, where the z p axis is parallel to the direction of light propagation, and the 1 / e 2 intensity level. The beam full width at z p = 3.8 mm must be < 250 μm for the exciting light to propagate past the approach-limiting edge without producing excessive light scattering.

Fig. 6
Fig. 6

Close up photographs of the (a) upright and (b) inverted aoTIRF configurations. The slightly red pencil of light inside the prisms is the 488 nm laser beam exciting autofluorescence.

Fig. 7
Fig. 7

The BFP microscopy emission pathway optical setup where OP is the object plane, OBJ is the objective, BFP is the back focal plane, TL is the tube lens with focal length f TL , RL is the chromatic and spherical aberrations corrected removable lens, and CCD is the image plane for the camera.

Fig. 8
Fig. 8

(a) Sequential images of 100 nm diameter fluorescent spheres under aoTIRF illumination in the upright microscope configuration. The images are 20 ms exposures of the CCD camera to light but are separated in time by 59 ms . (b) Sequential images of 100 nm diameter fluorescent spheres under aoTIRF illumination in the inverted microscope configuration. Exposure time was 100 ms on the EMCCD camera and frames are separated in time by 135 ms . (c) Sequential images of 40 nm diameter spheres diffusing in bulk solution under epi-illumination and observed in the upright microscope configuration. The images are 10 ms exposures of the CCD camera to light but are separated in time by 80 ms . Scale bars are shown for each panel.

Fig. 9
Fig. 9

The BFP image from a 100 nm diameter sphere emitting fluorescence excited by aoTIRF illumination (top half) and the pattern computed from a single emitting dipole lying 50 nm above the interface and pointing horizontally along the x-axis direction (bottom half). The arrow at the bottom of the figure designates the bright ring due to critical angle emission.

Fig. 10
Fig. 10

Fluorescence images of cardiac papillary muscle fibers. Fluorescence is detected from GFP tagged myosin regulatory light chain specifically replacing the native regulatory light chain on myosin cross bridges. Images in (a) and (b) compare the same sample but under epi-illumination and aoTIRF illumination. (c) is from another cardiac papillary fiber under oTIRF. All images were taken with the inverted microscope and the scale bar applies to all panels.

Tables (1)

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Table 1 Event Statistics Observed for Nanospheres Diffusing in Water at Room Temperature

Equations (2)

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I = I 0 exp [ z d ] where     d = λ 4 π ( n g sin θ ) 2 n w 2 ,
F = F 0 exp [ z 0 d ] ,

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