Abstract

We demonstrate subwavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fluorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel. Confocal-supercritical angular fluorescence microscopy would be a powerful tool to achieve high-resolution surface imaging, especially for membrane imaging in biological samples.

© 2014 Optical Society of America

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2013 (1)

D. Axelrod, Biophys. J. 104, 1401 (2013).
[CrossRef]

2012 (1)

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

2011 (2)

2010 (1)

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, Phys. Rev. Lett. 105, 108103 (2010).
[CrossRef]

2009 (1)

2008 (1)

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

2007 (1)

2006 (2)

J. P. Overington, B. Al-Lazikani, and A. L. Hopkins, Nat. Rev. Drug Discov. 5, 993 (2006).
[CrossRef]

A. L. Mattheyses and D. Axelrod, J. Biomed. Opt. 11, 014006 (2006).
[CrossRef]

2004 (2)

2003 (1)

T. Ruckstuhl, M. Rankl, and S. Seeger, Biosens. Bioelectron. 18, 1193 (2003).
[CrossRef]

2001 (1)

D. Axelrod, J. Biomed. Opt. 6, 6 (2001).
[CrossRef]

2000 (1)

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Al-Lazikani, B.

J. P. Overington, B. Al-Lazikani, and A. L. Hopkins, Nat. Rev. Drug Discov. 5, 993 (2006).
[CrossRef]

Axelrod, D.

D. Axelrod, Biophys. J. 104, 1401 (2013).
[CrossRef]

A. L. Mattheyses and D. Axelrod, J. Biomed. Opt. 11, 014006 (2006).
[CrossRef]

D. Axelrod, J. Biomed. Opt. 6, 6 (2001).
[CrossRef]

Balaa, K.

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, Opt. Lett. 36, 3051 (2011).
[CrossRef]

Barroca, T.

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, Opt. Lett. 36, 3051 (2011).
[CrossRef]

Chon, J. W.

Chung, E.

Delahaye, J.

Enderlein, J.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Fort, E.

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, Opt. Lett. 36, 3051 (2011).
[CrossRef]

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

Grésillon, S.

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

Gu, M.

Hopkins, A. L.

J. P. Overington, B. Al-Lazikani, and A. L. Hopkins, Nat. Rev. Drug Discov. 5, 993 (2006).
[CrossRef]

Jung, S.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Kano, H.

Kim, Y.-H.

Lévêque-Fort, S.

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort, and E. Fort, Opt. Lett. 36, 3051 (2011).
[CrossRef]

Mattheyses, A. L.

A. L. Mattheyses and D. Axelrod, J. Biomed. Opt. 11, 014006 (2006).
[CrossRef]

Overington, J. P.

J. P. Overington, B. Al-Lazikani, and A. L. Hopkins, Nat. Rev. Drug Discov. 5, 993 (2006).
[CrossRef]

Rankl, M.

T. Ruckstuhl, M. Rankl, and S. Seeger, Biosens. Bioelectron. 18, 1193 (2003).
[CrossRef]

Ruckstuhl, T.

T. Ruckstuhl, D. Verdes, C. Winterflood, and S. Seeger, Opt. Express 19, 6836 (2011).
[CrossRef]

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, Phys. Rev. Lett. 105, 108103 (2010).
[CrossRef]

T. Ruckstuhl and D. Verdes, Opt. Express 12, 4246 (2004).
[CrossRef]

T. Ruckstuhl, M. Rankl, and S. Seeger, Biosens. Bioelectron. 18, 1193 (2003).
[CrossRef]

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Seeger, S.

T. Ruckstuhl, D. Verdes, C. Winterflood, and S. Seeger, Opt. Express 19, 6836 (2011).
[CrossRef]

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, Phys. Rev. Lett. 105, 108103 (2010).
[CrossRef]

T. Ruckstuhl, M. Rankl, and S. Seeger, Biosens. Bioelectron. 18, 1193 (2003).
[CrossRef]

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Sheppard, C. J.

So, P. T.

Tang, W. T.

Terakado, G.

Verdes, D.

Watanabe, K.

Winterflood, C.

Winterflood, C. M.

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, Phys. Rev. Lett. 105, 108103 (2010).
[CrossRef]

Anal. Chem. (1)

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, Anal. Chem. 72, 2117 (2000).
[CrossRef]

Appl. Opt. (2)

Biophys. J. (1)

D. Axelrod, Biophys. J. 104, 1401 (2013).
[CrossRef]

Biosens. Bioelectron. (1)

T. Ruckstuhl, M. Rankl, and S. Seeger, Biosens. Bioelectron. 18, 1193 (2003).
[CrossRef]

J. Biomed. Opt. (2)

D. Axelrod, J. Biomed. Opt. 6, 6 (2001).
[CrossRef]

A. L. Mattheyses and D. Axelrod, J. Biomed. Opt. 11, 014006 (2006).
[CrossRef]

J. Phys. D (1)

E. Fort and S. Grésillon, J. Phys. D 41, 013001 (2008).
[CrossRef]

Nat. Rev. Drug Discov. (1)

J. P. Overington, B. Al-Lazikani, and A. L. Hopkins, Nat. Rev. Drug Discov. 5, 993 (2006).
[CrossRef]

Opt. Express (3)

Opt. Lett. (1)

Phys. Rev. Lett. (2)

T. Barroca, K. Balaa, S. Lévêque-Fort, and E. Fort, Phys. Rev. Lett. 108, 218101 (2012).
[CrossRef]

C. M. Winterflood, T. Ruckstuhl, D. Verdes, and S. Seeger, Phys. Rev. Lett. 105, 108103 (2010).
[CrossRef]

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Figures (5)

Fig. 1.
Fig. 1.

Simplified schematic of a confocal-SAF microscope with an amplitude mask placed in a conjugate plane of the objective’s back focal plane.

Fig. 2.
Fig. 2.

(a) Detected fluorescence intensity for an axial scan of the excitation focus in the confocal and SAF modes. The test sample was a 1 mM solution of rhodamine 6G in ethanol with nonnegligible fluorescence in the volume. Intensity counts were obtained for each z step for 30 s and normalized with respect to the maximum detected value along the axis. (b) An image of the conjugated back focal plane for a solution of 100 nm beads on the surface, and (c) same as (b) but with a central mask.

Fig. 3.
Fig. 3.

(a), (b) Images of the surface planes of a 5 μm fluorescent bead in confocal and SAF detection modes, respectively. (c), (d) Corresponding images 2.5 μm deep inside the bead imaging its center. The image stack was normalized to the maximum intensity on the surface plane in its corresponding detection mode. (e) Depicts the intensity decay for emitters located at various depths away from the interface extracted from the radial intensity profile of the surface plane.

Fig. 4.
Fig. 4.

Normalized lateral profile of (a) 100 nm beads and (b) 5 μm beads on the coverslip in an aqueous solution.

Fig. 5.
Fig. 5.

Actin filaments in fixed CHO cells tagged with ATTO 488 dye in (a) confocal detection and (b) confocal-SAF detection. (Scale bar 1μm, field of view: 20μm×20μm.)

Equations (1)

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Image(r,z)=(PSF(r,z)Object)×MCE(z)×Q,

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