Abstract

Latex microspheres are used as a simple and low-cost means to achieve three axis electromagnetic confinement below the standard diffraction limit. We demonstrate their use to enhance the fluorescence fluctuation detection of single molecules. Compared to confocal microscopy with high numerical aperture, we monitor a detection volume reduction of one order of magnitude below the diffraction limit together with a 5-fold gain in the fluorescence rate per molecule. This offers new opportunities for a broad range of applications in biophotonics, plasmonics, optical data storage and ultramicroscopy.

© 2008 Optical Society of America

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References

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  1. H. G. Craighead, "Future lab-on-a-chip technologies for interrogating individual molecules," Nature (London) 442, 387-393 (2006).
    [CrossRef] [PubMed]
  2. E. Fort and S. Gr???esillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008).
    [CrossRef]
  3. H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
    [CrossRef] [PubMed]
  4. X. Li, Z. Chen, A. Taflove, and V. Backman, "Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets," Opt. Express 13, 526-533 (2005).
    [CrossRef] [PubMed]
  5. P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, "Direct imaging of photonic nanojets," Opt. Express 16, 6930-6940 (2008).
    [CrossRef] [PubMed]
  6. A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef]
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    [CrossRef]
  11. A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
    [CrossRef] [PubMed]
  12. T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
    [CrossRef] [PubMed]
  13. J. Wenger, D. G???erard, A. Aouani, and H. Rigneault, "Disposable Microscope Objective Lenses for Fluorescence Correlation Spectroscopy using Latex Microspheres," Anal. Chem. 80, 6800-6804 (2008).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2008

E. Fort and S. Gr???esillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008).
[CrossRef]

P. Ferrand, J. Wenger, M. Pianta, H. Rigneault, A. Devilez, B. Stout, N. Bonod, and E. Popov, "Direct imaging of photonic nanojets," Opt. Express 16, 6930-6940 (2008).
[CrossRef] [PubMed]

J. Wenger, D. G???erard, A. Aouani, and H. Rigneault, "Disposable Microscope Objective Lenses for Fluorescence Correlation Spectroscopy using Latex Microspheres," Anal. Chem. 80, 6800-6804 (2008).
[CrossRef] [PubMed]

J. Wenger,  et al, "Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures," Opt. Express 16, 3008-3020 (2008).
[CrossRef] [PubMed]

2007

2006

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
[CrossRef] [PubMed]

H. G. Craighead, "Future lab-on-a-chip technologies for interrogating individual molecules," Nature (London) 442, 387-393 (2006).
[CrossRef] [PubMed]

2005

2000

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

1999

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Akiyama, H.

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Baba, M.

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Backman, V.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

X. Li, Z. Chen, A. Taflove, and V. Backman, "Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets," Opt. Express 13, 526-533 (2005).
[CrossRef] [PubMed]

Blom, H.

H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
[CrossRef] [PubMed]

Bonod, N.

Chen, Z.

Cr???egut, O.

Craighead, H. G.

H. G. Craighead, "Future lab-on-a-chip technologies for interrogating individual molecules," Nature (London) 442, 387-393 (2006).
[CrossRef] [PubMed]

Devilez, A.

Eggeling, C.

H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
[CrossRef] [PubMed]

Enderlein, J.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

Ferrand, P.

Fort, E.

E. Fort and S. Gr???esillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008).
[CrossRef]

Gennerich, A.

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

Gr???esillon, S.

E. Fort and S. Gr???esillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008).
[CrossRef]

Haacke, S.

Heifetz, A.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

Hirlimann, C.

Huang, K.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

Jung, S.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

Kastrup, L.

H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
[CrossRef] [PubMed]

Koyama, K.

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Lecler, S.

Lecong, N.

Li, X.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

X. Li, Z. Chen, A. Taflove, and V. Backman, "Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets," Opt. Express 13, 526-533 (2005).
[CrossRef] [PubMed]

Neviere, M.

Pianta, M.

Popov, E.

Rehspringer, J.-L.

Rigneault, H.

Ruckstuhl, T.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

Sahakian, A. V.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

Schild, D.

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

Seeger, S.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

Stout, B.

Suemoto, T.

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Taflove, A.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

X. Li, Z. Chen, A. Taflove, and V. Backman, "Optical analysis of nanoparticles via enhanced backscattering facilitated by 3-D photonic nanojets," Opt. Express 13, 526-533 (2005).
[CrossRef] [PubMed]

Wenger, J.

Yoshita, M.

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Anal. Chem.

T. Ruckstuhl, J. Enderlein, S. Jung, and S. Seeger, "Forbidden Light Detection from Single Molecules," Anal. Chem. 72, 2117-2123 (2000).
[CrossRef] [PubMed]

J. Wenger, D. G???erard, A. Aouani, and H. Rigneault, "Disposable Microscope Objective Lenses for Fluorescence Correlation Spectroscopy using Latex Microspheres," Anal. Chem. 80, 6800-6804 (2008).
[CrossRef] [PubMed]

Appl. Phys. Lett.

A. Heifetz, K. Huang, A. V. Sahakian, X. Li, A. Taflove, and V. Backman, "Experimental confirmation of backscattering enhancement induced by a photonic jet," Appl. Phys. Lett. 89, 221118 (2006).
[CrossRef]

K. Koyama, M. Yoshita, M. Baba, T. Suemoto, and H. Akiyama, "High collection efficiency in fluorescence microscopy with a solid immersion lens," Appl. Phys. Lett. 75, 1667-1669 (1999).
[CrossRef]

Biophys. J.

A. Gennerich and D. Schild, "Fluorescence Correlation Spectroscopy in Small Cytosolic Compartments Depends Critically on the Diffusion Model used," Biophys. J. 79, 3294-3306 (2000).
[CrossRef] [PubMed]

Curr. Pharm. Biotechnol.

H. Blom, L. Kastrup, and C. Eggeling, "Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes," Curr. Pharm. Biotechnol. 7, 51-66 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

J. Phys. D: Appl. Phys.

E. Fort and S. Gr???esillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008).
[CrossRef]

Nature (London)

H. G. Craighead, "Future lab-on-a-chip technologies for interrogating individual molecules," Nature (London) 442, 387-393 (2006).
[CrossRef] [PubMed]

Opt. Express

Other

R. Rigler and E. S. Elson, Fluorescence correlation spectroscopy : theory and applications (Springer, Berlin, 2001).
[CrossRef]

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Figures (4)

Fig. 1.
Fig. 1.

(a) Microsphere configuration for single molecule detection enhancement. Numerical simulations are computed without (b) and (c) in the presence of a 2 µm sphere illuminated by a focused Gaussian beam at λ = 633 nm with NA = 1.2 (factor of 2 between adjacent contours). Cuts along the horizontal and vertical axis at best focus outside the sphere are given in (d) and (e). Dashed lines correspond to a Gaussian illumination without the sphere, solid lines are for the resulting beam with the microsphere.

Fig. 2.
Fig. 2.

(a) Schematics of the experimental set-up. (b) Correlation functions recorded in free solution and on a 2 µm sphere, using the same A647 solution (crosses: raw data ; lines: numerical fits). Analysis based on Eq. (1) yields for the free solution : N = 21.3, τd = 71 µs, nT = 0.80, τ b T = 1.9 μ s , , CRM = 14.5 kHz, and for the 2 µm sphere : N = 2.06, τd = 22.6 µs, n T = 0.76 , τ b T = 0.8 μ s , CRM = 62.0 kHz. The insert shows a snapshot of the raw total fluorescence signal.

Fig. 3.
Fig. 3.

Observation volume reduction (blue diamonds) and CRM enhancement (red circles) versus the focus position with respect to the 2 µm sphere.

Fig. 4.
Fig. 4.

CRM enhancement and observation volume reduction vs. diameter.

Equations (1)

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g ( 2 ) τ = 1 + 1 N [ 1 + n T exp ( τ / τ b T ) ] ( 1 + τ / τ d ) 1 + s 2 τ / τ d ,

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