Abstract

We introduce a full-field fluorescence imaging technique with axial confinement of about 100nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity. It can be implemented on any epifluorescence microscope with a commercial high numerical aperture objective and offers a real-time surface imaging capability. This technique is of particular interest for live cell membrane and adhesion studies. Using human embryonic kidney cells, we show that one can observe simultaneously the surface and in-depth cell phenomena.

© 2011 Optical Society of America

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

2008

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

2007

2006

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

2005

M. Oheim and F. Schapper, J. Phys. D 38, R185 (2005).
[CrossRef]

2004

2003

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

2001

D. Axelrod, Traffic 2, 764 (2001).
[CrossRef] [PubMed]

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

2000

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

1998

M. S. Bretscher and C. Aguado-Velasco, Curr. Opin. Cell Biol. 10, 537 (1998).
[CrossRef] [PubMed]

1986

1959

B. Richards and E. Wolf, Proc. R. Soc. Lond. A 253, 358 (1959).
[CrossRef]

Aguado-Velasco, C.

M. S. Bretscher and C. Aguado-Velasco, Curr. Opin. Cell Biol. 10, 537 (1998).
[CrossRef] [PubMed]

Axelrod, D.

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

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

D. Axelrod, Traffic 2, 764 (2001).
[CrossRef] [PubMed]

Bretscher, M. S.

M. S. Bretscher and C. Aguado-Velasco, Curr. Opin. Cell Biol. 10, 537 (1998).
[CrossRef] [PubMed]

Chung, E.

Enderlein, J.

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

Fort, E.

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]

Jung, S.

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

Kim, Y-H.

Mahajan, V. N.

Mattheyses, A. L.

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

Oheim, M.

M. Oheim and F. Schapper, J. Phys. D 38, R185 (2005).
[CrossRef]

Olivo-Marin, J. C.

Rankl, M.

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

Richards, B.

B. Richards and E. Wolf, Proc. R. Soc. Lond. A 253, 358 (1959).
[CrossRef]

Rivolta, C.

Ruckstuhl, T.

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

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

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

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

Schapper, F.

M. Oheim and F. Schapper, J. Phys. D 38, R185 (2005).
[CrossRef]

Seeger, S.

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

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

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

Sheppard, C. J. R.

So, P. T. C.

Tang, W. T.

Verdes, D.

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

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

Winterflood, C. M.

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

Wolf, E.

B. Richards and E. Wolf, Proc. R. Soc. Lond. A 253, 358 (1959).
[CrossRef]

Zerubia, J.

Zhang, B.

Anal. Chem.

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

Appl. Opt.

Biosens. Bioelectron.

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

Curr. Opin. Cell Biol.

M. S. Bretscher and C. Aguado-Velasco, Curr. Opin. Cell Biol. 10, 537 (1998).
[CrossRef] [PubMed]

J. Biomed. Opt.

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

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

J. Opt. Soc. Am. A

J. Phys. D

M. Oheim and F. Schapper, J. Phys. D 38, R185 (2005).
[CrossRef]

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

Opt. Express

Phys. Rev. Lett.

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

Proc. R. Soc. Lond. A

B. Richards and E. Wolf, Proc. R. Soc. Lond. A 253, 358 (1959).
[CrossRef]

Traffic

D. Axelrod, Traffic 2, 764 (2001).
[CrossRef] [PubMed]

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Figures (4)

Fig. 1
Fig. 1

(a) Polar plots of the emission pattern for an isotropically oriented fluorophore positioned at the glass/water interface. (b) Normalized MDE versus fluorophore/ interface distance for SAF and for various TIRF angles.

Fig. 2
Fig. 2

Schematic of the experimental setup for multimodal real-time imaging with two parallel channels for the epifluorescence and F 2 -SAF images. DM, dichroic mirror; L1, lens1; L2, lens2; L3, lens3; L4, lens4; Exc. filter, excitation filter; Em. filter, emission filter.

Fig. 3
Fig. 3

(a) Normalized PSF profiles in UAF + SAF and F 2 -SAF: model (dashed lines) and experiment (smooth lines). (b), (c) Experimental PSF images in UAF + SAF and F 2 -SAF respectively; associated calculated images are shown in (d) and (e).

Fig. 4
Fig. 4

Images of HEK cells in (a)  UAF + SAF mode and in (b)  F 2 -SAF mode.

Equations (1)

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MDE ( d ) = Γ exc ( d ) × Q Y ( d ) × MCE ( d ) ,

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