Abstract

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

© 2011 OSA

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  1. E. H. Synge, “A suggested method for extending the microscopic resolution into the ultramicroscopic region,” Philos. Mag. 6, 356–362 (1928).
  2. D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
    [CrossRef]
  3. A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
    [CrossRef]
  4. T. Hirschfeld, “Total reflection fluorescence,” Can. Spectrosc. 10, 128 (1965).
  5. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
    [CrossRef] [PubMed]
  6. H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
    [CrossRef] [PubMed]
  7. W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power,” J. Opt. Soc. Am. A 67(12), 1607–1625 (1977).
    [CrossRef]
  8. E. D. Hellen and D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4(3), 337–350 (1987).
    [CrossRef]
  9. J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999).
    [CrossRef]
  10. T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
    [CrossRef] [PubMed]
  11. T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12(18), 4246–4254 (2004).
    [CrossRef] [PubMed]
  12. D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
    [CrossRef] [PubMed]
  13. J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008).
    [CrossRef]
  14. T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29(6), 569–571 (2004).
    [CrossRef] [PubMed]
  15. K. Hassler, M. Leutenegger, P. Rigler, R. Rao, R. Rigler, M. Gösch, and T. Lasser, “Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low background and high count-rate per molecule,” Opt. Express 13(19), 7415–7423 (2005).
    [CrossRef] [PubMed]
  16. N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007).
    [CrossRef] [PubMed]
  17. C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
    [CrossRef] [PubMed]
  18. B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253(1274), 358–379 (1959).
    [CrossRef]

2010 (1)

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

2008 (1)

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

2007 (2)

D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
[CrossRef] [PubMed]

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

2005 (2)

2004 (2)

2000 (1)

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

1999 (1)

1987 (1)

1984 (2)

D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
[CrossRef]

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

1981 (1)

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

1977 (1)

W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power,” J. Opt. Soc. Am. A 67(12), 1607–1625 (1977).
[CrossRef]

1965 (1)

T. Hirschfeld, “Total reflection fluorescence,” Can. Spectrosc. 10, 128 (1965).

1959 (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253(1274), 358–379 (1959).
[CrossRef]

1928 (1)

E. H. Synge, “A suggested method for extending the microscopic resolution into the ultramicroscopic region,” Philos. Mag. 6, 356–362 (1928).

Axelrod, D.

E. D. Hellen and D. Axelrod, “Fluorescence emission at dielectric and metal-film interfaces,” J. Opt. Soc. Am. B 4(3), 337–350 (1987).
[CrossRef]

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

Denk, W.

D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
[CrossRef]

Enderlein, J.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999).
[CrossRef]

Gösch, M.

Harootunian, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

Hassler, K.

Hellen, E. D.

Hirschfeld, T.

T. Hirschfeld, “Total reflection fluorescence,” Can. Spectrosc. 10, 128 (1965).

Isaacson, M.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

Jung, S.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

Kunz, R. E.

W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power,” J. Opt. Soc. Am. A 67(12), 1607–1625 (1977).
[CrossRef]

Lanz, M.

D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
[CrossRef]

Lasser, T.

Leutenegger, M.

Lewis, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

Lukosz, W.

W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power,” J. Opt. Soc. Am. A 67(12), 1607–1625 (1977).
[CrossRef]

Muray, A.

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

Pohl, D. W.

D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
[CrossRef]

Rao, R.

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253(1274), 358–379 (1959).
[CrossRef]

Ries, J.

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

Rigler, P.

Rigler, R.

Ruckstuhl, T.

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

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

D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
[CrossRef] [PubMed]

T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29(6), 569–571 (2004).
[CrossRef] [PubMed]

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

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999).
[CrossRef]

Schneckenburger, H.

H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
[CrossRef] [PubMed]

Schwille, P.

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

Seeger, S.

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
[CrossRef] [PubMed]

T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29(6), 569–571 (2004).
[CrossRef] [PubMed]

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999).
[CrossRef]

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]

Synge, E. H.

E. H. Synge, “A suggested method for extending the microscopic resolution into the ultramicroscopic region,” Philos. Mag. 6, 356–362 (1928).

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]

Verdes, D.

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

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

D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
[CrossRef] [PubMed]

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

Winterflood, C. M.

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253(1274), 358–379 (1959).
[CrossRef]

Anal. Chem. (1)

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

D. W. Pohl, W. Denk, and M. Lanz, “Optical Stethoscopy: Image Recording with Resolution λ/20,” Appl. Phys. Lett. 44(7), 651–653 (1984).
[CrossRef]

Biophys. J. (1)

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

Can. Spectrosc. (1)

T. Hirschfeld, “Total reflection fluorescence,” Can. Spectrosc. 10, 128 (1965).

Curr. Opin. Biotechnol. (1)

H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
[CrossRef] [PubMed]

J. Biomed. Opt. (1)

D. Verdes, T. Ruckstuhl, and S. Seeger, “Parallel two-channel near- and far-field fluorescence microscopy,” J. Biomed. Opt. 12(3), 034012 (2007).
[CrossRef] [PubMed]

J. Cell Biol. (1)

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

J. Opt. Soc. Am. A (1)

W. Lukosz and R. E. Kunz, “Light emission by magnetic and electric dipoles close to a plane interface. I. Total radiated power,” J. Opt. Soc. Am. A 67(12), 1607–1625 (1977).
[CrossRef]

J. Opt. Soc. Am. B (1)

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]

Opt. Express (2)

Opt. Lett. (1)

Philos. Mag. (1)

E. H. Synge, “A suggested method for extending the microscopic resolution into the ultramicroscopic region,” Philos. Mag. 6, 356–362 (1928).

Phys. Rev. Lett. (1)

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, “Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy,” Phys. Rev. Lett. 105(10), 108103 (2010).
[CrossRef] [PubMed]

Proc. R. Soc. Lond. A (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A 253(1274), 358–379 (1959).
[CrossRef]

Ultramicroscopy (1)

A. Lewis, M. Isaacson, A. Harootunian, and A. Muray, “Development of a 500 Å spatial resolution light microscope: I. light is efficiently transmitted through λ/16 diameter apertures,” Ultramicroscopy 13(3), 227–231 (1984).
[CrossRef]

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Figures (8)

Fig. 1
Fig. 1

Angular distribution of the radiant intensity of isotropically oriented dipole emitters into the glass halfspace. The plots were calculated for emitters in water with a distance to the glass surface of z = 0 (solid), z = λ/20 (dashed), z = λ/5 (dash-dotted) and of z = λ (dotted), with the emission wavelength λ. The inset shows a polar plot of the radiant intensity into the entire space for emitters with z = 0. SAF is the proportion of the fluorescence emitted above 61°, the critical angle of the water/glass interface.

Fig. 2
Fig. 2

Photographs and schematic of the developed microscope objective. Due to the reflection at the parabola the lowest collected angle comes to lie at the outside of the ejected annular ring of fluorescence and can be increased straightforwardly by means of a circular aperture.

Fig. 3
Fig. 3

Z-dependent fluorescence collection efficiency of the outer collector calculated for the angular cut-off at 62° (solid), 66° (dashed) and 70° (dash-dotted) for (A) aqueous solution (n 1 = 1.333, θc = 61°) and for (B) a biological cell (n 1 = 1.38, θc = 65°). A cut-off angle below the critical angle leads to a loss of the axial confinement of the detection volume. An isotropic dipole orientation was assumed for the calculations of the collection efficiencies.

Fig. 4
Fig. 4

Schematic of the optical setup.

Fig. 5
Fig. 5

Intensity distributions measured in the respective image planes of (A) SAF and (B) UAF. The circles indicate the 0.18 mm active area diameter of the SPADs.

Fig. 6
Fig. 6

Normalized point spread functions for UAF collection (upper) and for SAF collection (lower). (A) Scanned UAF image of a 36 nm diameter bead. Pixels: 78 nm × 78 nm. (B) Calculated lateral UAF-PSF at the interface and (C) along z. (D) Scanned SAF image the bead. (E) Calculated lateral SAF-PSF at the interface and (F) along z. Each image has an edge length of 2 μm.

Fig. 7
Fig. 7

Diffusion of fluorescent 36 nm diameter beads in water. Top: Raw data of the synchronized SAF and UAF intensities during the transition of two beads (left and right) through the excitation focus. The dashed horizontal line indicates the threshold intensity for the z-localization. Bottom: Z-position of the beads calculated from the data above for every 0.2 ms time bin with SAF and UAF intensities above the threshold.

Fig. 8
Fig. 8

Single molecule imaging and z-localization of IgG-Atto647N immobilized on a coverslip. (A) SAF image and (B) UAF image of a surface area of 13 μm × 13 μm. (C) Calculated z-positions indicated in nanometers. (D) Z-Localization histogram of molecules found on a larger surface area of 2000 μm2.

Equations (1)

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θ c = arcsin ( n 1 n 2 ) ,

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