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Virtual Journal for Biomedical Optics

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  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 6, Iss. 8 — Aug. 26, 2011
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Large molecular fluorescence enhancement by a nanoaperture with plasmonic corrugations

Heykel Aouani, Oussama Mahboub, Eloïse Devaux, Hervé Rigneault, Thomas W. Ebbesen, and Jérôme Wenger  »View Author Affiliations


Optics Express, Vol. 19, Issue 14, pp. 13056-13062 (2011)
http://dx.doi.org/10.1364/OE.19.013056


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Abstract

We investigate the influence of circular corrugations surrounding a central nanoaperture to further enhance the fluorescence count rate per emitter and control its emission directionality. Adding a single corrugation already allows to significantly increase the fluorescence signal as compared to a bare nanoaperture. A complete fluorescence characterization quantifies the excitation and emission gains contributing to the fluorescence enhancement process as the number of corrugations is increased.

© 2011 Optical Society of America

1. Introduction

In this paper, we focus on the influence of the number of circular corrugations surrounding a central nanoaperture to further enhance the fluorescence count rate per emitter. A complete fluorescence characterization combining correlation spectroscopy and lifetime measurements quantifies the excitation and emission gains contributing to the fluorescence enhancement as the number of corrugations is increased. By reciprocally coupling the far field radiation to the local energy inside the central aperture, the circular grating antenna leads to fluorescence enhancement factors significantly above those obtained with non-corrugated apertures.

2. Materials and methods

2.1. Corrugated nanoapertures fabrication

The preparation of the corrugated nanoapertures is based on two-step direct focused ion beam milling (FIB) [11

11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

]. First, the glass substrate is coated with a thin chromium adhesion layer and a 50 nm thick gold layer to make the substrate conductive. Periodic concentric corrugations are milled into the glass substrate using optimized design parameters derived from reference [10

10. O. Mahboub, S. Carretero Palacios, C. Genet, F. J. Garcia-Vidal, S. G. Rodrigo, L. Martin-Moreno, and T. W. Ebbesen, “Optimization of bulls eye structures for transmission enhancement,” Opt. Express 18, 11292–11299 (2010). [CrossRef] [PubMed]

]. The groove period is 440 nm, width 200 nm, depth 65 nm, and there is an increasing number of corrugations up to 5 grooves. Two layers of gold (140 nm) and chromium (60 nm) are then deposited on top of the corrugated substrate to obtain the final gold thickness with a chromium layer on top [11

11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

]. Finally, a central aperture of 140 nm diameter is opened with FIB. This diameter is chosen to provide maximum fluorescence enhancement [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

]. Typical scanning electron microscopy (SEM) images of the fabricated samples are presented in Fig. 1.

Fig. 1 (a) Scanning electron microscope image of the fabricated nanoapertures with 1, 2, 3 and 5 corrugations. (b) and (c) Experimental configuration.

2.2. Fluorescence correlation spectroscopy to quantify the fluorescence enhancement

Fluorescence correlation spectroscopy (FCS) is a powerful tool to count the number of emitters in a defined observation volume. FCS records the temporal fluctuations of the fluorescence signal F(t), and computes its temporal correlation G(2)(τ) = 〈F(t).F (t + τ)〉/〈F(t)〉2, where 〈.〉 stands for time-averaging. Numerical analysis of the FCS data quantifies the average number of molecule N in the observation volume (full details on the FCS analysis can be found in [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

] and in the supporting information of [11

11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

], Fig. 2(a) presents a typical fluorescence correlation function and corresponding time trace). It is then straightforward to compute the fluorescence count rate per molecule CRM (the number of photons emitted by a molecule per second) as CRM = 〈F(t)〉/N. This enables an accurate measurement of fluorescence enhancement factors.

Fig. 2 (a) Typical fluorescence correlation function and fluorescence raw time trace in the case of an aperture with three corrugations and 400 μW excitation power. (b) Fluorescence count rate per molecule CRM versus excitation power for the different samples. (c) Parameters of the numerical fits in (b). (d) Fluorescence enhancement factors relative to the open solution, in the weak excitation regime. The line is a guide to the eyes.

To distinguish the relative contributions of the excitation and emission gains in the overall fluorescence enhancement factor, we have developed a specific procedure [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

]. Briefly, the fluorescence CRM is measured by FCS while increasing the excitation power Ie. This set of data is fitted by the standard expression of the fluorescence count rate per molecule: CRM = AIe/(1 + Ie/Is), where A is a constant proportional to the molecular absorption cross-section, emission rate and setup collection efficiency and Is is the saturation power. In the saturation regime (IeIs), CRM = AIs and the fluorescence signal does not depend anymore on the excitation. The fluorescence enhancement at saturation, which corresponds to the the ratio between the products AIs of the fitting parameters, is equivalent to the emission gain ηem brought by the antenna. This emission gain ηem is expressed as the product of the radiative rate enhancement ηrad times the collection efficiency enhancement ηκ: ηem = ηradηκ. We stress that there is no need to reach practically the saturation regime, the numerical fits in Fig. 2(b) suffice to determine the saturation intensity Is and the CRM asymptotic limit AIs.

In the weak excitation regime (IeIs), the fluorescence enhancement equals ηF = CRMaperture/CRMsolution = Aaperture/Asolution. This can be specified as ηF = ηκηϕηexc, [1

1. L. Novotny and N. H. van Hulst, “Antennas for light,” Nat. Photonics , 5, 83–90 (2011). [CrossRef]

] where ηκ is the collection efficiency enhancement, ηexc is the excitation intensity enhancement and ηϕ = ηradtot is the quantum efficiency enhancement (ratio of radiative rate enhancement ηrad to the modification of the total fluorescence decay rate ηtot). The alteration in the total fluorescence decay rate ηtot is determined separately by fluorescence lifetime measurements using the standard Time-Correlated Single Photon Counting (TCSPC) method, which is detailed in Ref. [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

]. To quantify the excitation intensity gain ηexc, we rewrite the fluorescence enhancement as ηF = ηexcηemtot to make apparent the emission gain ηem. The separate knowledge of ηF, ηem and ηtot finally quantifies the excitation intensity gain ηexc. This procedure separates the excitation, emission, and decay rate contributions. Please note that the emission gain ηem contains a term proportional to the setup collection efficiency. Therefore, the ratio ηemtot = ηκηϕ is greater than the quantum yield enhancement of the fluorophore [11

11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

]. More details about this procedure can be found in reference [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

].

2.3. Experimental setup

The experimental setup (Fig, 1) uses a 0.5 NA water-immersion objective (Zeiss Neofluar). This moderate numerical aperture was chosen to realize a spot diameter in the focal plane of about 1.5 μm, in order to cover the circular corrugations surrounding the apertures. All experiments are performed on Alexa Fluor 647 molecules (A647, Invitrogen, Carlsbad CA, absorption / emission peaks at 650 and 672 nm) diluted in a standard water-based phosphate buffered saline (PBS) solution. For FCS, excitation is performed with a 632.8 nm CW linearly polarized laser beam, while a picosecond laser diode at 636 nm is used for fluorescence lifetime measurements. All excitation power measurements are performed at the entrance port of the confocal microscope. The backward-emitted fluorescence is detected by avalanche photodiodes with 670±20 nm bandpass filters. For FCS, the fluorescence temporal fluctuations F(t) are recorded by a hardware correlator (ALV6000, ALV GmbH, Langen). For fluorescence lifetime measurements, the photodiode signal is sent to a fast time-correlation counting module (PicoHarp300, Picoquant GmbH, Berlin) [12

12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

].

To analyse the angular distribution of the fluorescence radiation, we image the fluorescence intensity in the back focal plane of a 1.2 NA water immersion objective [4

4. A. Curto, G. Volpe, T.H. Taminiau, M. Kreuzer, R. Quidant, and N. F. Van Hulst, “Unidirectional Emission of a Quantum Dot Coupled to a Nanoantenna,” Science , 329, 930–933 (2010). [CrossRef] [PubMed]

, 11

11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

]. By property of the back focal plane (Fourier plane), the radial coordinate in these images represents the numerical aperture nsin(θ), where the medium refractive index is n = 1.33 and θ is the emission polar angle. The back focal plane images thus represent the radiation pattern for different angular directions from the antenna.

3. Experimental results and discussion

To quantify the influence of the circular corrugations, we compute the fluorescence enhancement in the low excitation limit ηF = CRMaperture/CRMsolution = Aaperture/Asolution (Fig. 2(d)). We observe a growing behavior of the enhancement factors as the number of corrugations is increased from 0 (ηF = 14.5) up to 5 (ηF = 106). These results deserve several comments. Firstly, the 106-fold enhancement by the aperture with 5 corrugations is remarkably the highest fluorescence enhancement reported to date for Alexa Fluor 647 molecules in solution. Secondly, we do observe a kind of saturation effect of the enhancement factor brought by adding a supplementary corrugation when the number of corrugations exceeds 3. This is a direct consequence of (i) the limited excitation spot to about 1.5 μm diameter, which brings less energy to the more distant corrugations, and (ii) increased plasmon propagation losses as the corrugation distance is increased to the central aperture. Milling 3 corrugations appears as a good compromise between fluorescence enhancement and nanofabrication complexity in the case of our setup. Thirdly, a single corrugation already provides a 3.5 times higher enhancement factor (ηF = 51.5) as compared to a bare aperture. This constitutes the first experimental observation of the effect numerically predicted in [13

13. N. Bonod, E. Popov, D. Gérard, J. Wenger, and H. Rigneault, “Field enhancement in a circular aperture surrounded by a single channel groove,” Opt. Express 16, 2276–2287 (2008). [CrossRef] [PubMed]

], and stands in good agreement with the values inferred in this reference.

Fig. 3 Excitation ηexc, emission ηem and total decay rate ηtot gains contributing to the global fluorescence enhancement ηF = ηexcηemtot.

To quantify the fluorescence emission angular distribution, we record the fluorescence intensity images in the back focal plane of a 1.2 NA microscope objective. Figure 4(a) presents our experimental results. For the non-corrugated aperture (N=0), the image contains a single disk representing the maximum collection angle at 64°, while for all corrugated apertures, the image contains an additional bright spot centered on the optical axis. This set of data is analyzed to display the different radiation patterns in the polar graphs in Fig. 4(b). The emission from a non-corrugated aperture is mostly limited by our collection NA, while the emission from the corrugated apertures can be tuned to high directionality in the direction normal to the sample by simply increasing the number of grooves. Starting with a single groove (N=1), the emission half width at half maximum (HWHM) is 37°, which already shows a nice control of the emission directivity. Further increasing the number of grooves narrows the angular distribution to 14° with N=2 and 10° with N=3.

Fig. 4 (a) Fluorescence intensity distribution in the back focal plane of a 1.2 NA objective for a single nanoaperture with an increasing number N of periodic corrugations. (b) Angular radiation patterns corresponding to the images in (a).

4. Conclusion

We quantify the dependence of the fluorescence enhancement per molecule on the number of circular corrugations surrounding a nanoaperture. The circular grating antenna increases both the excitation and emission rates of single emitters diffusing inside the central aperture, leading to fluorescence enhancement factors significantly above those obtained with bare apertures. Additionally, we demonstrate efficient single molecule detection in solution with a simple low NA lens. We show that a single groove milled around a nanoaperture already provides a supplementary 3.5-fold increase in the fluorescence enhancement as compared to a bare nanoaperture, realizing the first experimental observation of the effect numerically predicted in [13

13. N. Bonod, E. Popov, D. Gérard, J. Wenger, and H. Rigneault, “Field enhancement in a circular aperture surrounded by a single channel groove,” Opt. Express 16, 2276–2287 (2008). [CrossRef] [PubMed]

]. These results and the many opportunities for further optimization [10

10. O. Mahboub, S. Carretero Palacios, C. Genet, F. J. Garcia-Vidal, S. G. Rodrigo, L. Martin-Moreno, and T. W. Ebbesen, “Optimization of bulls eye structures for transmission enhancement,” Opt. Express 18, 11292–11299 (2010). [CrossRef] [PubMed]

, 13

13. N. Bonod, E. Popov, D. Gérard, J. Wenger, and H. Rigneault, “Field enhancement in a circular aperture surrounded by a single channel groove,” Opt. Express 16, 2276–2287 (2008). [CrossRef] [PubMed]

] open promising routes for ultimate control of the emission from a single quantum emitter [1

1. L. Novotny and N. H. van Hulst, “Antennas for light,” Nat. Photonics , 5, 83–90 (2011). [CrossRef]

].

Acknowledgments

This research is partly funded by the European Research Council under contract 227577 Plasmonics, and by the Provence-Alpes-Côte d’Azur Region.

References and links

1.

L. Novotny and N. H. van Hulst, “Antennas for light,” Nat. Photonics , 5, 83–90 (2011). [CrossRef]

2.

Y. Fu and J. R. Lakowicz, “Modification of single molecule fluorescence near metallic nanostructures,” Laser Photon. Rev. 3, 221–232 (2009). [CrossRef]

3.

A. Kinkhabwala, Z. F. Yu, S. H. Fan, Y. Avlasevich, K. Mullen, and W. E. Moerner, “Large Single-Molecule Fluorescence Enhancements Produced by a Bowtie Nanoantenna,” Nat. Photonics 3, 654–657 (2009). [CrossRef]

4.

A. Curto, G. Volpe, T.H. Taminiau, M. Kreuzer, R. Quidant, and N. F. Van Hulst, “Unidirectional Emission of a Quantum Dot Coupled to a Nanoantenna,” Science , 329, 930–933 (2010). [CrossRef] [PubMed]

5.

F. J. Garcia-Vidal, L. Martin-Moreno, T. W. Ebbesen, and L. Kuipers, “Light passing through subwavelength apertures,” Rev. Mod. Phys. , 82, 729–787 (2010). [CrossRef]

6.

H. J. Lezec, A. Degiron, E. Devaux, R. A. Linke, L. Martin-Moreno, F. J. Garcia-Vidal, and T. W. Ebbesen, “Beaming Light from a Subwavelength Aperture,” Science , 297, 820–822 (2002). [CrossRef] [PubMed]

7.

E. Laux, C. Genet, T. Skauli, and T. W. Ebbesen, “Plasmonic photon sorters for spectral and polarimetric imaging,” Nat. Photonics 2, 161–164 (2008). [CrossRef]

8.

A. Nahata, R. A. Linke, T. Ishi, and K. Ohashi, “Enhanced nonlinear optical conversion from a periodically nanostructured metal film,” Opt. Lett. 28, 423–425 (2003). [CrossRef] [PubMed]

9.

T. Ishi, J. Fujikata, K. Makita, T. Baba, and K. Ohashi, “Si Nano-Photodiode with a Surface Plasmon Antenna,” Jap. J. Appl. Phys. 44, L364L366 (2005). [CrossRef]

10.

O. Mahboub, S. Carretero Palacios, C. Genet, F. J. Garcia-Vidal, S. G. Rodrigo, L. Martin-Moreno, and T. W. Ebbesen, “Optimization of bulls eye structures for transmission enhancement,” Opt. Express 18, 11292–11299 (2010). [CrossRef] [PubMed]

11.

H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. 11, 637–644 (2011). [CrossRef] [PubMed]

12.

J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]

13.

N. Bonod, E. Popov, D. Gérard, J. Wenger, and H. Rigneault, “Field enhancement in a circular aperture surrounded by a single channel groove,” Opt. Express 16, 2276–2287 (2008). [CrossRef] [PubMed]

OCIS Codes
(050.1220) Diffraction and gratings : Apertures
(050.2770) Diffraction and gratings : Gratings
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(240.6680) Optics at surfaces : Surface plasmons
(050.6624) Diffraction and gratings : Subwavelength structures

ToC Category:
Diffraction and Gratings

History
Original Manuscript: January 24, 2011
Revised Manuscript: February 25, 2011
Manuscript Accepted: February 27, 2011
Published: June 22, 2011

Virtual Issues
Vol. 6, Iss. 8 Virtual Journal for Biomedical Optics

Citation
Heykel Aouani, Oussama Mahboub, Eloïse Devaux, Hervé Rigneault, Thomas W. Ebbesen, and Jerome Wenger, "Large molecular fluorescence enhancement by a nanoaperture with plasmonic corrugations," Opt. Express 19, 13056-13062 (2011)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-19-14-13056


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References

  1. L. Novotny, and N. H. van Hulst, “Antennas for light,” Nat. Photonics 5, 83–90 (2011). [CrossRef]
  2. Y. Fu, and J. R. Lakowicz, “Modification of single molecule fluorescence near metallic nanostructures,” Laser Photon. Rev. 3, 221–232 (2009). [CrossRef]
  3. A. Kinkhabwala, Z. F. Yu, S. H. Fan, Y. Avlasevich, K. Mullen, and W. E. Moerner, “Large Single-Molecule Fluorescence Enhancements Produced by a Bowtie Nanoantenna,” Nat. Photonics 3, 654–657 (2009). [CrossRef]
  4. A. Curto, G. Volpe, T. H. Taminiau, M. Kreuzer, R. Quidant, and N. F. Van Hulst, “Unidirectional Emission of a Quantum Dot Coupled to a Nanoantenna,” Science 329, 930–933 (2010). [CrossRef] [PubMed]
  5. F. J. Garcia-Vidal, L. Martin-Moreno, T. W. Ebbesen, and L. Kuipers, “Light passing through subwavelength apertures,” Rev. Mod. Phys. 82, 729–787 (2010). [CrossRef]
  6. H. J. Lezec, A. Degiron, E. Devaux, R. A. Linke, L. Martin-Moreno, F. J. Garcia-Vidal, and T. W. Ebbesen, “Beaming Light from a Subwavelength Aperture,” Science 297, 820–822 (2002). [CrossRef] [PubMed]
  7. E. Laux, C. Genet, T. Skauli, and T. W. Ebbesen, “Plasmonic photon sorters for spectral and polarimetric imaging,” Nat. Photonics 2, 161–164 (2008). [CrossRef]
  8. A. Nahata, R. A. Linke, T. Ishi, and K. Ohashi, “Enhanced nonlinear optical conversion from a periodically nanostructured metal film,” Opt. Lett. 28, 423–425 (2003). [CrossRef] [PubMed]
  9. T. Ishi, J. Fujikata, K. Makita, T. Baba, and K. Ohashi, “Si Nano-Photodiode with a Surface Plasmon Antenna,” Jap. J. Appl. Phys. 44, L364-L366 (2005). [CrossRef]
  10. O. Mahboub, S. Carretero Palacios, C. Genet, F. J. Garcia-Vidal, S. G. Rodrigo, L. Martin-Moreno, and T. W. Ebbesen, “Optimization of bulls eye structures for transmission enhancement,” Opt. Express 18, 11292–11299 (2010). [CrossRef] [PubMed]
  11. H. Aouani, O. Mahboub, N. Bonod, E. Devaux, E. Popov, H. Rigneault, T. W. Ebbesen, and J. Wenger, “Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations,” Nano Lett. , 637–644 (2011). [CrossRef] [PubMed]
  12. J. Wenger, D. Gérard, J. Dintinger, O. Mahboub, N. Bonod, E. Popov, T. W. Ebbesen, and H. Rigneault, “Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures,” Opt. Express 16, 3008–3020 (2008). [CrossRef] [PubMed]
  13. N. Bonod, E. Popov, D. Gérard, J. Wenger, and H. Rigneault, “Field enhancement in a circular aperture surrounded by a single channel groove,” Opt. Express 16, 2276–2287 (2008). [CrossRef] [PubMed]

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