Simultaneous near-field and far-field fluorescence microscopy of single molecules

doi:10.1364/OE.19.006836

Simultaneous near-field and far-field fluorescence microscopy of single molecules

Thomas Ruckstuhl, Dorinel Verdes, Christian M. Winterflood, and Stefan Seeger »View Author Affiliations

Optics Express, Vol. 19, Issue 7, pp. 6836-6844 (2011)
http://dx.doi.org/10.1364/OE.19.006836

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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.

OCIS Codes
(080.3620) Geometric optics : Lens system design
(180.2520) Microscopy : Fluorescence microscopy
(180.4243) Microscopy : Near-field microscopy

ToC Category:
Microscopy

History
Original Manuscript: February 14, 2011
Revised Manuscript: March 15, 2011
Manuscript Accepted: March 16, 2011
Published: March 24, 2011

Virtual Issues
Vol. 6, Iss. 4 Virtual Journal for Biomedical Optics

Citation
Thomas Ruckstuhl, Dorinel Verdes, Christian M. Winterflood, and Stefan Seeger, "Simultaneous near-field and far-field fluorescence microscopy of single molecules," Opt. Express 19, 6836-6844 (2011)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-19-7-6836

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References

E. H. Synge, “A suggested method for extending the microscopic resolution into the ultramicroscopic region,” Philos. Mag. 6, 356–362 (1928).
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]
T. Hirschfeld, “Total reflection fluorescence,” Can. Spectrosc. 10, 128 (1965).
D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981). [CrossRef] [PubMed]
H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005). [CrossRef] [PubMed]
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]
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]
J. Enderlein, T. Ruckstuhl, and S. Seeger, “Highly efficient optical detection of surface-generated fluorescence,” Appl. Opt. 38(4), 724–732 (1999). [CrossRef]
T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, “Forbidden light detection from single molecules,” Anal. Chem. 72(9), 2117–2123 (2000). [CrossRef] [PubMed]
T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12(18), 4246–4254 (2004). [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]
J. Ries, T. Ruckstuhl, D. Verdes, and P. Schwille, “Supercritical angle fluorescence correlation spectroscopy,” Biophys. J. 94(1), 221–229 (2008). [CrossRef]
T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29(6), 569–571 (2004). [CrossRef] [PubMed]
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]
N. L. Thompson and B. L. Steele, “Total internal reflection with fluorescence correlation spectroscopy,” Nat. Protoc. 2(4), 878–890 (2007). [CrossRef] [PubMed]
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]
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]

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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]

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Simultaneous near-field and far-field fluorescence microscopy of single molecules