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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 3, Iss. 4 — Apr. 23, 2008
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Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures

Jérôme Wenger, Davy Gérard, José Dintinger, Oussama Mahboub, Nicolas Bonod, Evgeny Popov, Thomas W. Ebbesen, and Hervé Rigneault  »View Author Affiliations


Optics Express, Vol. 16, Issue 5, pp. 3008-3020 (2008)
http://dx.doi.org/10.1364/OE.16.003008


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Abstract

We detail the role of single nanometric apertures milled in a gold film to enhance the fluorescence emission of Alexa Fluor 647 molecules. Combining fluorescence correlation spectroscopy and lifetime measurements, we determine the respective contributions of excitation and emission in the observed enhanced fluorescence. We characterize a broad range of nanoaperture diameters from 80 to 310 nm, and highlight the link between the fluorescence enhancement and the local photonic density of states. These results are of great interest to increase the effectiveness of fluorescence-based single molecule detection and to understand the interaction between a quantum emitter and a nanometric metal structure.

© 2008 Optical Society of America

1. Introduction

The fluorescence emission of a single molecule can be enhanced by properly tailoring its photonic environment, leading to new opportunities for single molecule detection. The environment can affect the fluorescence emission in three ways: (i) by locally enhancing the excitation intensity, (ii) by increasing the emitter’s radiative rate and quantum efficiency, and (iii) by modifying its radiation pattern, towards a higher emission directionality to the detectors. Determining the influence of these processes is a crucial issue to characterize nanodevices for enhanced fluorescence, which has been a topic of great interest for the last decade [1

1. W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt. 45, 661–699 (1998). [CrossRef]

, 2

2. J. R. Lakowicz, “Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission,” Anal. Biochem. 337, 171–194 (2005). [CrossRef] [PubMed]

, 3

3. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008). [CrossRef]

].

In the growing field of nanostructure-enhanced fluorescence, much attention is currently devoted to metal structures, where surface plasmons can play an additional role [2

2. J. R. Lakowicz, “Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission,” Anal. Biochem. 337, 171–194 (2005). [CrossRef] [PubMed]

, 3

3. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008). [CrossRef]

]. For instance, recent studies involve metallic nanoparticles [4

4. P. Anger, P. Bharadwaj, and L. Novotny, “Enhancement and Quenching of Single-Molecule Fluorescence,” Phys. Rev. Lett. 96, 113002 (2006). [CrossRef] [PubMed]

, 5

5. S. Kühn, U. Håkanson, L. Rogobete, and V. Sandoghdar, “Enhancement of Single-Molecule Fluorescence using a Gold Nanoparticle as an Optical Nanoantenna,” Phys. Rev. Lett. 97, 017402 (2006). [CrossRef] [PubMed]

, 6

6. J. Zhang, Y. Fu, M. H. Chowdhury, and J. R. Lakowicz, “Metal-Enhanced Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect between Metal Particles,” Nano Lett. 7, 2101–2107 (2007). [CrossRef] [PubMed]

, 7

7. S. Gerber, F. Reil, U. Hohenester, T. Schlagenhaufen, J. R. Krenn, and A. Leitner, “Tailoring light emission properties of fluorophores by coupling to resonance-tuned metallic nanostructures,” Phys. Rev. B 75, 073404 (2007). [CrossRef]

, 8

8. Y. Zhang, K. Aslan, M. J. R. Previte, and C. D. Geddes, “Metal-enhanced fluorescence: Surface plasmons can radiate a fluorophore’s structured emission,” Appl. Phys. Lett. 90, 053107 (2007). [CrossRef]

], core-shell particles [9

9. F. Tam, G. P. Goodrich, B. R. Johnson, and N. J. Halas, “Plasmonic Enhancement of Molecular Fluorescence,” Nano Lett. 7, 496–501 (2007). [CrossRef] [PubMed]

], thin films [10

10. J. Enderlein and T. Ruckstuhl, “The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection,” Opt. Express 13, 8855–8865 (2005). [CrossRef] [PubMed]

], nanoantennas [11

11. J. N. Farahani, D. W. Pohl, H.-J. Eisler, and B. Hecht, “Single Quantum Dot Coupled to a Scanning Optical Antenna: A Tunable Superemitter,” Phys. Rev. Lett. 95, 017402 (2005). [CrossRef] [PubMed]

, 12

12. O. L. Muskens, V. Giannini, J. A. Sanchez-Gil, and J. Gomez Rivas, “Strong Enhancement of the Radiative Decay Rate of Emitters by Single Plasmonic Nanoantennas,” Nano Lett. 7, 2871–2875 (2007). [CrossRef] [PubMed]

], nanoporous gold [13

13. J. S. Biteen, D. Pacifici, N. S. Lewis, and H. A. Atwater, “Enhanced Radiative Emission Rate and Quantum Efficiency in Coupled Silicon Nanocrystal-Nanostructured Gold Emitters,” Nano Lett. 5, 1768–1773 (2005). [CrossRef] [PubMed]

], nanopockets [14

14. G. L. Liu, J. Kim, and L. P. Lee, “Fluorescence enhancement of quantum dots enclosed in Au nanopockets with subwavelength aperture,” Appl. Phys. Lett. 89, 241118 (2006). [CrossRef]

], metallic gratings [15

15. Y.-J. Hung, I. I. Smolyaninov, C. C. Davis, and H.-C. Wu, “Fluorescence enhancement by surface gratings,” Opt. Express 14, 10825–10830 (2006). [CrossRef] [PubMed]

, 16

16. G. Sun, J. B. Khurgin, and R. A. Soref, “Practicable enhancement of spontaneous emission using surface plasmons,” Appl. Phys. Lett. 90, 111107 (2007). [CrossRef]

], nanoaperture arrays [17

17. Y. Liu and S. Blair, “Fluorescence enhancement from an array of subwavelength metal apertures,” Opt. Lett. 28, 507–509 (2003). [CrossRef] [PubMed]

, 18

18. A. G. Brolo, S. C. Kwok, M. D. Cooper, M. G. Moffitt, C.-W Wang, R. Gordon, J. Riordon, and K. L. Kavanagh, “Surface Plasmon-Quantum Dot Coupling from Arrays of Nanoholes,” J. Phys. Chem. B 110, 8307–8313 (2006). [CrossRef] [PubMed]

, 19

19. J. H. Kim and P. J. Moyer, “Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity,” Appl. Phys. Lett. 90, 131111 (2007). [CrossRef]

], and single nanoapertures [20

20. U. C. Fischer, “Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence,” J. Opt. Soc. Am. B 3, 1239–1244 (1986). [CrossRef]

, 21

21. H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005). [CrossRef] [PubMed]

]. For all these geometries, determining the specific influence of a nanostructure on the fluorescence emission remains a challenging task, as the detected signal results from a product of excitation and emission processes. Excitation depends on the interaction between the driving field and the nanostructure, while at moderate optical powers, the emission efficiency is set by the balance of radiative and non-radiative decays and the modification of the radiation pattern.

In spite of their conceptual simplicity, nanoapertures bear appealing properties to increase the effectiveness of fluorescence-based single-molecule detection [24

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

, 25

25. J. T. Mannion and H. G. Craighead, “Nanofluidic Structures for Single Biomolecule Fluorescent Detection,” Biopolymers 85, 131–143 (2006). [CrossRef] [PubMed]

, 26

26. C. Genet and T. W. Ebbesen, “Light in tiny holes,” Nature (London) 445, 39–46 (2007). [CrossRef] [PubMed]

]. Thanks to these devices, a large range of biologic processes can be efficiently monitored with single molecule resolution at micromolar concentrations [27

27. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003). [CrossRef] [PubMed]

, 28

28. K. T. Samiee, M. Foquet, L. Guo, E. C. Cox, and H. G. Craighead, “Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides,” Biophys. J. 88, 2145–2153 (2005). [CrossRef]

, 29

29. M. Leutenegger, M. Gösch, A. Perentes, P. Hoffmann, O. J. F. Martin, and T. Lasser, “Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture,” Opt. Express 14, 956–969 (2006). [CrossRef] [PubMed]

]. The use of nanoaperture for biophotonics applications can also be extended to the investigation of live cell membranes [30

30. J. Wenger, F. Conchonaud, J. Dintinger, L. Wawrezinieck, T.W. Ebbesen, H. Rigneault, D. Marguet, and P. F. Lenne, “Diffusion Analysis within Single Nanometric Apertures Reveals the Ultrafine Cell Membrane Organization,” Biophys. J. 92, 913–919 (2007). [CrossRef]

] and duallabels cross-correlation studies [31

31. J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, “Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations,” Opt. Express 14, 12206–12216 (2006). [CrossRef] [PubMed]

]. Therefore, understanding the fluorescence alteration in a nanoaperture is of practical importance for high-efficiency single-molecule analysis.

The paper is outlined as follows. In Section 2, we describe the procedure used to characterize the different photokinetic rates. Section 3 presents the experimental methods used throughout the paper. Results on the fluorescence detection rates and lifetimes are presented in Section 4 for aperture diameters ranging from 80 to 310 nm. In Section 5, we combine the experimental results to detail the fluorescence photokinetics alteration and assess the respective role of excitation and emission enhancements in the fluorescence process. Finally, we summarize our conclusions in Section 6.

2. Fluorescence characterization procedure

To introduce our characterization procedure in a few words, we implement FCS to reliably quantify the detected number of molecules and the fluorescence count rate per molecule CRM, which is recorded as the excitation power Ie is raised. Global analysis of the CRM vs. Ie curve, combined to lifetime measurements using time correlated single photon counting (TCSPC) alongside the FCS setup, gives the relative contribution of emission and excitation gains in the overall fluorescence enhancement, in a similar trend as in references [32

32. J. Widengren, R. Rigler, and U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4, 255–258 (1994). [CrossRef]

, 33

33. J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995). [CrossRef]

].We point out that this characterization procedure combining FCS and TCSPC can be straightforwardly extended to other types of plasmonic nanostructures.

Fig. 1. (a) Schematic view of the experimental setup combining FCS and TCSPC. (b) Nanoaperture configuration. (c) Notations used to describe the molecular transition rates.

We introduce the fluorescence enhancement ηF in a nanoaperture as the ratio of the detected fluorescence rate per molecule in the aperture CRMaper and in open solution CRMsol at a fixed excitation power, that is ηF=CRMaper/CRMsol. To understand the physical origin of the increase in the fluorescence brightness already reported in [23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

], we begin by expressing the fluorescence rate per molecule CRM. Throughout this paper, we treat Alexa Fluor 647 as a three energy levels system, as illustrated in Fig. 1(c). S0 denotes the ground state, S1 the excited singlet state and D is a dark (non-fluorescent) state that accounts for both triplet state and non-fluorescing isomerized form. Although the molecular structure of Alexa Fluor 647 has not been made available yet, the FCS curves and fluorescence lifetimes clearly indicate that it has a carbocyanine structure, with a quantum yield in water solution of about 30% [34

34. V. Buschmann, K. D Weston, and M. Sauer, “Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes,” Bioconjugate Chem. 14, 195–204 (2003). [CrossRef]

]. To the aim of the study reported here, and as far as the fluorescence brightness is concerned, this model will be sufficient. A more detailed description of the photokinetics of cyanine 5 dyes can be found in [35

35. J. Widengren and P. Schwille, “Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000). [CrossRef]

].

The photokinetic rates are noted as follows, and are summarized in appendix A: σIe stands for the excitation rate, where σ denotes the excitation cross-section and Ie the excitation intensity. krad and knrad are the rate constants for radiative emission and non-radiative deexcitation from S1 to the ground state. kisc and kd are the rate constants for inter-system crossing to the dark state and relaxation to the ground state respectively. The total deexcitation rate from the excited singlet state S1 is noted as ktot=1/τtot, where τtot is the excited state lifetime.

Under steady-state conditions, the fluorescence rate per molecule CRM is given by [36

36. C. Zander, J. Enderlein, and R. A. Keller (Eds), Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002). [CrossRef]

]

CRM=κϕσIe1+IeIs
(1)

where κ is the light collection efficiency, ϕ=krad/ktot the quantum yield and Is=ktotσ11+kisckd is the saturation intensity.

To ease the understanding of our measurements, we introduce the emission rate kem=κkrad, which denotes the effectively detected radiation rate by our instrument. Both radiative rate and collection efficiency are accounted into a single factor kem. Eq. (1) then rewrites

CRM=kemktotσIe1+IeIs
(2)

In the low excitation regime IeIs, Eq. (2) reduces to

CRMlow=kemktotσIe(IeIs)
(3)

which indicates that the fluorescence rate per molecule is proportional to the excitation intensity and to the ratio of the detected emission rate to the overall decay rate. The fluorescence enhancement ηF can therefore be expressed as

ηF,low=CRMaperCRMsol=ηkemηktotηIe(IeIs)
(4)

where ηkem=kem-aper/kem-sol, ηktot=ktot-aper/ktot-sol and ηIeIe-aperIe-sol are the enhancements in the collected emission rate, total decay rate and excitation rate.

In the saturation regime IeIs, Eq. (2) reduces to

CRMsat=kemktotσIs=kem1+kisckd(IeIs)
(5)

which indicates that the fluorescence rate per molecule at saturation is determined by the collected emission rate and the ratio kisc/kd. It is important to note that kisc/kd is proportional to the relative population of the dark state D once saturation is reached [32

32. J. Widengren, R. Rigler, and U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4, 255–258 (1994). [CrossRef]

, 33

33. J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995). [CrossRef]

]. We verified experimentally that the dark state fraction at fluorescence saturation was similar for all the nanoaperture diameters, as for the open solution.We therefore make the reasonable assumption that at fluorescence saturation the ratio kisc/kd is a constant set by the Alexa Fluor 647 properties, and that it is independent of the photonic environment. While computing the ratios of the fluorescence rates at saturation ηF,sat, the term 11+kisckd in Eq. (5) thus disappears, and one ends up with the simple expression

ηF,sat=ηkem(IeIs)
(6)

To characterize the fluorescence photokinetics enhancement induced by a single nanoaperture we will thus perform the following procedure:

  • The fluorescence rates per molecule CRM are measured by FCS for increasing excitation powers in open solution and in single nanoapertures.
  • The data points for CRM versus Ie are fitted according to Eq. (2). The ratio to the open solution gives ηF versus Ie. From the numerical fits, we infer the fluorescence enhancement at the limit below saturation ηF,low and at saturation ηF,sat.
  • According to Eq. (6), the value of ηF,sat at saturation equals the emission rate enhancement ηkem.
  • The fluorescence decay rate ktot is characterized by pulsed time-correlated measurements. The ratio of the results in the nanoaperture to the open solution gives ηktot.
  • According to Eq. (4), the excitation rate enhancement is obtained as ηIeF,low ηktotkem.

As we will show below, this procedure turns out to be very efficient in discriminating the contributions of excitation and emission to the overall fluorescence process. The limit of this method is that all the presented results account for spatial averaging over all the possible molecular orientations and positions inside the analyzed volume. There is no sensitivity to individual molecular trajectories or dipole orientations, but one ends up directly with global figures to characterize the (spatially averaged) emitted fluorescence. Besides, it is very difficult to reveal the physics underneath the emission rate kem=κkrad enhancement. Distinguishing between the contributions of the radiative rate krad and the collection efficiency κ remains a challenge, mainly because of the intrinsic difficulty to reliably measure a collection efficiency [22

22. J. Wenger, B. Cluzel, J. Dintinger, N. Bonod, A.- L. Fehrembach, E. Popov, P.-F. Lenne, T. W. Ebbesen, and H. Rigneault, “Radiative and Nonradiative Photokinetics Alteration Inside a Single Metallic Nanometric Aperture,” J. Phys. Chem. C 111, 11469–11474 (2007). [CrossRef]

, 23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

]. Last, the fluorescence enhancement factors are spectrally averaged within the fluorescence bandpass detection window.

3. Materials and methods

Our experimental set-up is based on an inverted microscope with a NA=1.2 water-immersion objective, allowing single aperture studies (Fig. 1). It combines on the same setup FCS and time-correlated lifetime measurements facilities, used to determine the complete photokinetics alteration in a nanoaperture following the procedure derived in section 2. We emphasize that for all experiments, a droplet of solution containing Alexa-Fluor 647 molecules is deposited on top of the aperture sample. The droplet acts as a reservoir of molecules, that are constantly diffusing inside the aperture, which strongly limits photobleaching effects.

3.1. Nanoapertures

Opaque gold films (thickness 200 nm) were coated on conventional microscope coverslips (thickness 150 µm) by thermal evaporation. A 15 nm thick chromium layer ensures adhesion between the gold film and the glass substrate. Circular apertures with diameters ranging from 80 nm to 310 nm were then directly milled by focused ion beam (FEI Strata DB235).

3.2. FCS measurements and analysis

For FCS measurements, the excitation is set to a CW HeNe laser operating at 633 nm. After a 50 µm confocal pinhole conjugated to the sample plane, the detection is performed by two avalanche photodiodes (Perkin-Elmer SPCM-AQR-13) with 670±20 nm fluorescence band-pass filters. The fluorescence intensity fluctuations are analyzed by cross-correlating the signal of each photodiode with a ALV6000 hardware correlator. Each individual FCS measurement was obtained by averaging 5 runs of 10 s duration.

As an illustration, Fig. 2 presents typical correlograms g (2)(τ) recorded in a 120 nm nanoaperture for three different excitation powers. The analysis of this FCS data relies on a numerical fit based on a three dimensional Brownian diffusion model [23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

, 36

36. C. Zander, J. Enderlein, and R. A. Keller (Eds), Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002). [CrossRef]

]:

g(2)(τ)=1+1N(1BF)2[1+nTexp(ττbT)]1(1+ττd)1+s2ττd
(7)

where N is the total number of molecules, 〈F〉 the total signal, 〈B〉 the background noise, nT the amplitude of the dark state population, τbT the dark state blinking time, τd the mean diffusion time and s the ratio of transversal to axial dimensions of the analysis volume. Numerical fit of the FCS data following Eq. (7) provides the average number of molecules N which is used to compute the fluorescence count rate per molecule CRM.

The background noise 〈B〉 originates mainly from the back-reflected laser light and from gold autofluorescence. At 40 µW excitation powers, it typically amounts to 〈B〉=3 kHz, which is almost negligible as compared to the count rates per molecule in the nanoapertures in the range 50–150 kHz (Fig. 3). In the configuration of Fig. 1, the aperture subwavelength diameter and the 200 nm gold thickness provide strong electromagnetic confinement at the aperture bottom. Therefore, fluorescence contribution from the pool of molecules above the nanoaperture was shown to be negligible [21

21. H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005). [CrossRef] [PubMed]

].

Fig. 2. (a) Typical fluorescence autocorrelations in a 120 nm aperture (crosses, raw data) and numerical fits according to Eq. (7) (lines). (b) Snapshot of the raw fluorescence signal corresponding to (a).

3.3. TCSPC measurements and fluorescence lifetime analysis

To measure the fluorescence decay rate, we use the following procedure that takes into account the limited resolution of our setup. Careful analysis of the instrument response function (IRF) (shown later on Fig. 5(a)) reveals a double exponential decay: IRF(t)∝A 1 exp(-k 1 t)+A 2 exp(-k 2 t) with A 1=0.516, A 2=0.484, k 1=5.7 109 s-1 and k 2=20.7 109 s-1. The output signal O(t) of the TCSPC card corresponds to the convolution of the system IRF with the averaged fluorescence decay, which is assumed to be mono-exponential. Convolving a mono-exponential fluorescence decay with a double exponential IRF results in a triple exponential:

O(t)(A1+A2)exp(ktott)A1exp(k1t)A2exp(k2t)
(8)

where ktot is the molecular total deexcitation rate, and A 1, A 2, k 1 and k 2 are fixed parameters set by the IRF analysis. While analyzing the experimental decay curves, ktot is thus kept as the only free varying parameter. This procedure yields a fluorescence lifetime of 1.0 ns for Alexa Fluor 647 in open water solution, which corresponds well to the data in the literature [34

34. V. Buschmann, K. D Weston, and M. Sauer, “Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes,” Bioconjugate Chem. 14, 195–204 (2003). [CrossRef]

].

4. Experimental results

To get a global picture of the fluorescence photokinetics alterations in a single nanoaperture, we carried extensive FCS experiments for aperture diameters ranging from 80 up to 310 nm. The excitation power was increased from 25 to 500 µW (the upper limit was set to avoid damaging the sample and photobleaching the dyes). Each correlation function was analyzed to compute the average number of molecules N and fluorescence count rate per molecule CRM. Please note that special care has been taken to characterize the level of background noise and the dark state amplitude for each excitation power.

Fig. 3. Fluorescence rates per molecule CRM versus excitation power in open solution and in single nanoapertures. Circles are experimental data, lines are numerical fits using Eq. (2).
Fig. 4. (a) Fluorescence enhancement ηF derived from Fig. 3. (b) Fluorescence enhancement below saturation ηF,low (empty markers) and at saturation ηF,sat (filled markers) deduced from the numerical fits in Fig. 3 according to Eqs. (2), (4) and (6).
Fig. 5. (a) Normalized fluorescence decay traces measured in open solution (black dots) and in single nanoapertures. Dots are experimental data, lines are numerical fits following the procedure described in Sec. 3.3. The shorter decay trace (grey) is the overall instrument response function (IRF). (b) Fluorescence lifetime reduction versus the aperture diameter (as compared to open solution), deduced from the numerical fits in (a) using Eq. (8).

Figure 3 displays the evolution of the detected fluorescence rate per molecule CRM versus the excitation power for the different aperture diameters. Solid lines are numerical fits according to Eq. (2), which agree well to our experimental data. As the aperture diameter decreases, the fluorescence rate CRM increases before reaching an optimum for a diameter of 120 nm, which was previously assessed to give the largest fluorescence enhancement [23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

]. For smaller apertures (100 and 80 nm diameters), we show that CRM is decreased. Figure 3 also indicates that high count rates larger than 400,000 counts per second and per molecule are readily obtained in nanoapertures at high excitation power, while in open solution, the count rate saturates to values below 180,000 counts per second.

Count rates displayed on Fig. 3 were used to compute the corresponding fluorescence enhancement factors ηF=CRMaper/CRMsol. The results are displayed on Fig. 4(a) versus the excitation power, with an optimum diameter at 120 nm. Then, we use the numerical fits of CRM versus Ie according to Eqs. (2), (4) and (6) to infer the fluorescence enhancement well below saturation ηF,low and at saturation ηF,sat. For clarity, we present these figures versus the aperture diameter in Fig. 4(b). The lower value for ηF,sat as compared to ηF,low results from the respective influence of the excitation intensity enhancement and lifetime reduction, as indicated in Eqs. (4) and (6). We point out that the reported fluorescence enhancement factors account for spectral averaging over the 650–690 nm detection window. Spectral dependence of the fluorescence enhancement has been investigated in [23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

], and was shown to cover the entire dye emission spectrum with only slight spectral variations.

Along with the FCS measurements, for the same nanoaperture sample and the same Alexa Fluor 647 solution, we conducted TCSPC experiments to investigate the fluorescence lifetime alteration inside the nanoapertures. Figure 5(a) shows the measured fluorescence decay curves (dots) and their numerical fits (lines) for Alexa 647 molecules in single nanoapertures of diameters ranging from 80 up to 310 nm. As the aperture diameter is decreased, a clear reduction in the fluorescence lifetime is seen. To quantify this reduction, we fitted the data according to Eq. (8), and normalized the measured decay rate to the decay rate in open solution. This yields the ratio ηktot=ktot-aper/ktot-sol displayed on Fig. 5(b). ηktot increases as the aperture diameter is decreased, and as the metal comes closer to the average position of the molecules. We measure a clear lifetime reduction of about 3 for a 80 nm aperture.

5. Discussion

The different experimental results on Fig. 4 and 5 are now combined to estimate the alteration of the fluorescence photokinetics rates. We use the different steps described in section 2: according to Eq. (6), the fluorescence enhancement at saturation ηF,sat gives the emission rate enhancement ηkem, which denotes the gain in radiative rate and collection efficiency. Then, following Eq. (4), the excitation rate enhancement is obtained from the fluorescence enhancement below saturation and the lifetime reduction as ηIeF,low ηktotkem. For completeness, all these results are presented on one single figure for each aperture diameter (Fig. 6(a) to (d)). We also compute the ratio ηkemktot (Fig. 6(e)). This displays the gain in the factor Kem/ktot=κkrad/ktot=κϕ which is the product of the collection efficiency κ with the dye’s quantum yield ϕ. Last, to comment on these results, we display the propagation constant γ of the fundamental guided mode inside an infinitely long aperture as a function of the aperture diameter (Fig. 6(f)), the aperture being filled with water [23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

].

A general comment on Fig. 6 is that the nanoaperture affects both emission and excitation in the fluorescence process. Both effects contribute to the large fluorescence enhancement observed. Hereafter, we will relate these effects to the local density of states (LDOS) increase induced by the nanoaperture. The global consideration of the graphs on Fig. 6 leads to the introduction of three regions as indicated by the dashed vertical lines on Fig. 6. Region (1) is for aperture diameters below 100 nm, region (2) for diameters between 100 and 175 nm, and region (3) stands for diameters above 175 nm. These regions are only intended to guide the following discussion, there are obviously no strict borders between them.

Starting from region (3), the general trend is an increase in the photokinetic rates as the aperture diameter is decreased. To compare with the propagation constant γ (Fig. 6(f)) we point out that in this region, the real part of the propagation constant is large while the imaginary part is low, corresponding to a propagative excitation field. As the aperture diameter is decreased, the imaginary part of the propagation constant grows, leading to more electromagnetic confinement at the aperture entrance. This translates into an increase in the emission enhancement along with the excitation enhancement (Fig. 6(b) and (d)).

On the other hand, region (1) shows a decrease in the fluorescence enhancement as the aperture diameter is reduced. This is linked to a large increase in ktot (Fig. 6(c)), while at the same time, the emission and the excitation rates tend to decrease with the aperture diameter. Consequently, the apparent quantum yield kem/ktot decreases (Fig. 6(e)). These effects are strong evidences for fluorescence quenching, which we relate to a too close vicinity between the dyes and the metal. Let us also point out that region (1) corresponds to a large imaginary part of the propagation constant γ and thus to strongly evanescent fields inside the aperture and large losses. This contributes to the reduction in emission and excitation enhancement (Fig. 6(b) and (d)).

Fig. 6. Physical contributions to nanoaperture enhanced fluorescence, plotted versus the aperture diameter and normalized to the open solution case. (a) Fluorescence enhancement below saturation ηF,low, (b) Emission rate enhancement ηkem, (c) Lifetime reduction ηktot, (d) Excitation enhancement ηIe, (e) Ratio ηkemktot, (f) Propagation constant γ of the fundamental mode inside the aperture (solid line: real part, dashed line: imaginary part).

Last, region (2) appears as a trade-off between cases (1) and (3). This region is close to the cutoff of the fundamental mode that may propagate through the aperture and to the position where the group velocity is minimum (found for a diameter of 120 nm). These conditions lead to a maximum of the photonic density of states [1

1. W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt. 45, 661–699 (1998). [CrossRef]

], and therefore to a maximum in the emission and excitation rates (Fig. 6(b) and (d)). Moreover, the metal-dielectric interface set by the aperture may allow fluorescence energy transferred to a surface plasmon to be coupled out into the radiated field at the aperture edge, contributing to the emission [2

2. J. R. Lakowicz, “Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission,” Anal. Biochem. 337, 171–194 (2005). [CrossRef] [PubMed]

, 3

3. E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008). [CrossRef]

]. At the same time, we infer that the molecules stay (on average) far enough from the metal layer, so that the relative influence of quenching to the metal is limited. This is supported by a plateau in the apparent quantum yield (Fig. 6(e)).

Finally, we point out that our experimental results stand in good agreement with the numerical predictions based on the finite elements method [37

37. F. Mahdavi, Y. Liu, and S. Blair, “Modeling Fluorescence Enhancement from Metallic Nanocavities,” Plasmonics 2, 129–142 (2007). [CrossRef]

, 23

23. D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

] and differential theory [38

38. E. Popov, M. Nevière, J. Wenger, P.-F. Lenne, H. Rigneault, P. Chaumet, N. Bonod, J. Dintinger, and T. W. Ebbesen, “Field enhancement in single subwavelength apertures,” J. Opt. Soc. Am. A 23, 2342–2348 (2006). [CrossRef]

].

6. Conclusion

We have detailed the influence of single nanometric apertures on the fluorescence of Alexa Fluor 647 molecules, and determined the respective contributions of excitation and emission in the observed enhanced fluorescence. The procedure has been conducted for a broad range of nanoapertures with diameters from 80 to 310 nm, which allowed us to discriminate between different physical effects and relate the photokinetics enhancements to the local photonic density of states. Let us emphasize that the characterization procedure combining FCS and TCSPC can be straightforwardly extended to other types of plasmonic nanostructures.

Finally, this study shows that nanoapertures bear many interesting properties for biophotonics, such as light localization, sub-femtoliter observation volume, and an increase in the excitation and emission yield. These properties are of great interest to increase the effectiveness of fluorescence-based single molecule detection.

Appendix A: Fluorescence photokinetics notations

SymbolQuantity
S0 Molecular ground state
S1 Excited single state
DDark state
σIe Excitation rate to S1
σ Excitation cross-section
Ie Excitation intensity
krad Rate constant for radiative emission
knrad Rate constant for non-radiative deexcitation from S1 to the ground state
kisc Rate constant for inter-system crossing to the dark state D
kd Rate constant for relaxation from D to S0
ktot Total deexcitation rate from S1: ktot=krad+knrad+kisc
τtot Fluorescence lifetime τtot=1/ktot
N Average number of detected molecules
FAverage total fluorescence signal per second
CRM Fluorescence rate per molecule CRM=〈F〉/N
κ Collection efficiency
ϕ Quantum yield ϕ=krad/ktot
Is Saturation intensity Is=ktot/(σ(1+kisc/kd))
kem Effective emission rate kem=κ krad
ηF Fluorescence rate enhancement ηF=CRMaper/CRMsol
ηF,low Fluorescence rate enhancement below saturation (IeIs)
ηF,sat Fluorescence rate enhancement at saturation (IeIs)
ηkem Emission rate enhancement ηkem=kem-aper/kem-sol
ηktot Decay rate enhancement, lifetime reduction ηktot=ktot-aper/ ktot-sol
ηIe Excitation rate enhancement ηIe= σIe-aperIe-sol
ηκ Collection efficiency enhancement ηκ=κaper/κsol

Acknowledgments

The authors acknowledge stimulating discussions with S. Blair. This work has been funded by the French Agence Nationale de la Recherche under contract ANR-05-PNANO-035-01 “CO-EXUS” and ANR-07-NANO-006-03 “ANTARES”.

References and links

1.

W. L. Barnes, “Fluorescence near interfaces: the role of photonic mode density,” J. Mod. Opt. 45, 661–699 (1998). [CrossRef]

2.

J. R. Lakowicz, “Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission,” Anal. Biochem. 337, 171–194 (2005). [CrossRef] [PubMed]

3.

E. Fort and S. Grésillon, “Surface enhanced fluorescence,” J. Phys. D: Appl. Phys. 41, 013001 (2008). [CrossRef]

4.

P. Anger, P. Bharadwaj, and L. Novotny, “Enhancement and Quenching of Single-Molecule Fluorescence,” Phys. Rev. Lett. 96, 113002 (2006). [CrossRef] [PubMed]

5.

S. Kühn, U. Håkanson, L. Rogobete, and V. Sandoghdar, “Enhancement of Single-Molecule Fluorescence using a Gold Nanoparticle as an Optical Nanoantenna,” Phys. Rev. Lett. 97, 017402 (2006). [CrossRef] [PubMed]

6.

J. Zhang, Y. Fu, M. H. Chowdhury, and J. R. Lakowicz, “Metal-Enhanced Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect between Metal Particles,” Nano Lett. 7, 2101–2107 (2007). [CrossRef] [PubMed]

7.

S. Gerber, F. Reil, U. Hohenester, T. Schlagenhaufen, J. R. Krenn, and A. Leitner, “Tailoring light emission properties of fluorophores by coupling to resonance-tuned metallic nanostructures,” Phys. Rev. B 75, 073404 (2007). [CrossRef]

8.

Y. Zhang, K. Aslan, M. J. R. Previte, and C. D. Geddes, “Metal-enhanced fluorescence: Surface plasmons can radiate a fluorophore’s structured emission,” Appl. Phys. Lett. 90, 053107 (2007). [CrossRef]

9.

F. Tam, G. P. Goodrich, B. R. Johnson, and N. J. Halas, “Plasmonic Enhancement of Molecular Fluorescence,” Nano Lett. 7, 496–501 (2007). [CrossRef] [PubMed]

10.

J. Enderlein and T. Ruckstuhl, “The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection,” Opt. Express 13, 8855–8865 (2005). [CrossRef] [PubMed]

11.

J. N. Farahani, D. W. Pohl, H.-J. Eisler, and B. Hecht, “Single Quantum Dot Coupled to a Scanning Optical Antenna: A Tunable Superemitter,” Phys. Rev. Lett. 95, 017402 (2005). [CrossRef] [PubMed]

12.

O. L. Muskens, V. Giannini, J. A. Sanchez-Gil, and J. Gomez Rivas, “Strong Enhancement of the Radiative Decay Rate of Emitters by Single Plasmonic Nanoantennas,” Nano Lett. 7, 2871–2875 (2007). [CrossRef] [PubMed]

13.

J. S. Biteen, D. Pacifici, N. S. Lewis, and H. A. Atwater, “Enhanced Radiative Emission Rate and Quantum Efficiency in Coupled Silicon Nanocrystal-Nanostructured Gold Emitters,” Nano Lett. 5, 1768–1773 (2005). [CrossRef] [PubMed]

14.

G. L. Liu, J. Kim, and L. P. Lee, “Fluorescence enhancement of quantum dots enclosed in Au nanopockets with subwavelength aperture,” Appl. Phys. Lett. 89, 241118 (2006). [CrossRef]

15.

Y.-J. Hung, I. I. Smolyaninov, C. C. Davis, and H.-C. Wu, “Fluorescence enhancement by surface gratings,” Opt. Express 14, 10825–10830 (2006). [CrossRef] [PubMed]

16.

G. Sun, J. B. Khurgin, and R. A. Soref, “Practicable enhancement of spontaneous emission using surface plasmons,” Appl. Phys. Lett. 90, 111107 (2007). [CrossRef]

17.

Y. Liu and S. Blair, “Fluorescence enhancement from an array of subwavelength metal apertures,” Opt. Lett. 28, 507–509 (2003). [CrossRef] [PubMed]

18.

A. G. Brolo, S. C. Kwok, M. D. Cooper, M. G. Moffitt, C.-W Wang, R. Gordon, J. Riordon, and K. L. Kavanagh, “Surface Plasmon-Quantum Dot Coupling from Arrays of Nanoholes,” J. Phys. Chem. B 110, 8307–8313 (2006). [CrossRef] [PubMed]

19.

J. H. Kim and P. J. Moyer, “Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity,” Appl. Phys. Lett. 90, 131111 (2007). [CrossRef]

20.

U. C. Fischer, “Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence,” J. Opt. Soc. Am. B 3, 1239–1244 (1986). [CrossRef]

21.

H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen, and P.-F. Lenne, “Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures,” Phys. Rev. Lett. 95, 117401 (2005). [CrossRef] [PubMed]

22.

J. Wenger, B. Cluzel, J. Dintinger, N. Bonod, A.- L. Fehrembach, E. Popov, P.-F. Lenne, T. W. Ebbesen, and H. Rigneault, “Radiative and Nonradiative Photokinetics Alteration Inside a Single Metallic Nanometric Aperture,” J. Phys. Chem. C 111, 11469–11474 (2007). [CrossRef]

23.

D. Gérard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, “Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals,” Phys. Rev. B 77, 045413 (2008). [CrossRef]

24.

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

25.

J. T. Mannion and H. G. Craighead, “Nanofluidic Structures for Single Biomolecule Fluorescent Detection,” Biopolymers 85, 131–143 (2006). [CrossRef] [PubMed]

26.

C. Genet and T. W. Ebbesen, “Light in tiny holes,” Nature (London) 445, 39–46 (2007). [CrossRef] [PubMed]

27.

M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W Webb, “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations,” Science 299, 682–686 (2003). [CrossRef] [PubMed]

28.

K. T. Samiee, M. Foquet, L. Guo, E. C. Cox, and H. G. Craighead, “Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides,” Biophys. J. 88, 2145–2153 (2005). [CrossRef]

29.

M. Leutenegger, M. Gösch, A. Perentes, P. Hoffmann, O. J. F. Martin, and T. Lasser, “Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture,” Opt. Express 14, 956–969 (2006). [CrossRef] [PubMed]

30.

J. Wenger, F. Conchonaud, J. Dintinger, L. Wawrezinieck, T.W. Ebbesen, H. Rigneault, D. Marguet, and P. F. Lenne, “Diffusion Analysis within Single Nanometric Apertures Reveals the Ultrafine Cell Membrane Organization,” Biophys. J. 92, 913–919 (2007). [CrossRef]

31.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, “Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations,” Opt. Express 14, 12206–12216 (2006). [CrossRef] [PubMed]

32.

J. Widengren, R. Rigler, and U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluoresc. 4, 255–258 (1994). [CrossRef]

33.

J. Widengren, U. Mets, and R. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995). [CrossRef]

34.

V. Buschmann, K. D Weston, and M. Sauer, “Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes,” Bioconjugate Chem. 14, 195–204 (2003). [CrossRef]

35.

J. Widengren and P. Schwille, “Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy,” J. Phys. Chem. A 104, 6416–6428 (2000). [CrossRef]

36.

C. Zander, J. Enderlein, and R. A. Keller (Eds), Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002). [CrossRef]

37.

F. Mahdavi, Y. Liu, and S. Blair, “Modeling Fluorescence Enhancement from Metallic Nanocavities,” Plasmonics 2, 129–142 (2007). [CrossRef]

38.

E. Popov, M. Nevière, J. Wenger, P.-F. Lenne, H. Rigneault, P. Chaumet, N. Bonod, J. Dintinger, and T. W. Ebbesen, “Field enhancement in single subwavelength apertures,” J. Opt. Soc. Am. A 23, 2342–2348 (2006). [CrossRef]

OCIS Codes
(050.1220) Diffraction and gratings : Apertures
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence
(240.6680) Optics at surfaces : Surface plasmons
(160.4236) Materials : Nanomaterials

ToC Category:
Diffraction and Gratings

History
Original Manuscript: January 31, 2008
Revised Manuscript: February 15, 2008
Manuscript Accepted: February 15, 2008
Published: February 20, 2008

Virtual Issues
Vol. 3, Iss. 4 Virtual Journal for Biomedical Optics

Citation
Jérôme Wenger, Davy Gérard, José Dintinger, Oussama Mahboub, Nicolas Bonod, Evgeny Popov, Thomas W. Ebbesen, and Hervé Rigneault, "Emission and excitation contributions to enhanced single molecule fluorescence by gold nanometric apertures," Opt. Express 16, 3008-3020 (2008)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-16-5-3008


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References

  1. W. L. Barnes, "Fluorescence near interfaces: the role of photonic mode density," J. Mod. Opt. 45, 661-699 (1998). [CrossRef]
  2. J. R. Lakowicz, "Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission," Anal. Biochem. 337, 171-194 (2005). [CrossRef] [PubMed]
  3. E. Fort and S. Gresillon, "Surface enhanced fluorescence," J. Phys. D: Appl. Phys. 41, 013001 (2008). [CrossRef]
  4. P. Anger, P. Bharadwaj and L. Novotny, "Enhancement and Quenching of Single-Molecule Fluorescence," Phys. Rev. Lett. 96, 113002 (2006). [CrossRef] [PubMed]
  5. S. Kuhn, U. Hakanson, L. Rogobete and V. Sandoghdar, "Enhancement of Single-Molecule Fluorescence using a Gold Nanoparticle as an Optical Nanoantenna," Phys. Rev. Lett. 97, 017402 (2006). [CrossRef] [PubMed]
  6. J. Zhang, Y. Fu, M. H. Chowdhury, and J. R. Lakowicz, "Metal-Enhanced Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect between Metal Particles," Nano Lett. 7, 2101-2107 (2007). [CrossRef] [PubMed]
  7. S. Gerber, F. Reil, U. Hohenester, T. Schlagenhaufen, J. R. Krenn, and A. Leitner, "Tailoring light emission properties of fluorophores by coupling to resonance-tuned metallic nanostructures," Phys. Rev. B 75, 073404 (2007). [CrossRef]
  8. Y. Zhang, K. Aslan, M. J. R. Previte, and C. D. Geddes, "Metal-enhanced fluorescence: Surface plasmons can radiate a fluorophore’s structured emission," Appl. Phys. Lett. 90, 053107 (2007). [CrossRef]
  9. F. Tam, G. P. Goodrich, B. R. Johnson, and N. J. Halas, "Plasmonic Enhancement of Molecular Fluorescence," Nano Lett. 7, 496-501 (2007). [CrossRef] [PubMed]
  10. J. Enderlein and T. Ruckstuhl, "The efficiency of surface-plasmon coupled emission for sensitive fluorescence detection," Opt. Express 13, 8855-8865 (2005). [CrossRef] [PubMed]
  11. J. N. Farahani, D. W. Pohl, H.-J. Eisler, and B. Hecht, "Single Quantum Dot Coupled to a Scanning Optical Antenna: A Tunable Superemitter," Phys. Rev. Lett. 95, 017402 (2005). [CrossRef] [PubMed]
  12. O. L. Muskens, V. Giannini, J. A. Sanchez-Gil, and J. Gomez Rivas, "Strong Enhancement of the Radiative Decay Rate of Emitters by Single Plasmonic Nanoantennas," Nano Lett. 7, 2871-2875 (2007). [CrossRef] [PubMed]
  13. J. S. Biteen, D. Pacifici, N. S. Lewis and H. A. Atwater, "Enhanced Radiative Emission Rate and Quantum Efficiency in Coupled Silicon Nanocrystal-Nanostructured Gold Emitters," Nano Lett. 5, 1768-1773 (2005). [CrossRef] [PubMed]
  14. G. L. Liu, J. Kim, and L. P. Lee, "Fluorescence enhancement of quantum dots enclosed in Au nanopockets with subwavelength aperture," Appl. Phys. Lett. 89, 241118 (2006). [CrossRef]
  15. Y.-J. Hung, I. I. Smolyaninov, C. C. Davis and H.-C. Wu, "Fluorescence enhancement by surface gratings," Opt. Express 14, 10825-10830 (2006). [CrossRef] [PubMed]
  16. G. Sun, J. B. Khurgin and R. A. Soref, "Practicable enhancement of spontaneous emission using surface plasmons," Appl. Phys. Lett. 90, 111107 (2007). [CrossRef]
  17. Y. Liu and S. Blair, "Fluorescence enhancement from an array of subwavelength metal apertures," Opt. Lett. 28, 507-509 (2003). [CrossRef] [PubMed]
  18. A. G. Brolo, S. C. Kwok, M. D. Cooper, M. G. Moffitt, C.-W. Wang, R. Gordon, J. Riordon, and K. L. Kavanagh, "Surface Plasmon-Quantum Dot Coupling from Arrays of Nanoholes," J. Phys. Chem. B 110, 8307-8313 (2006). [CrossRef] [PubMed]
  19. J. H. Kim and P. J. Moyer, "Laser-induced fluorescence within subwavelength metallic arrays of nanoholes indicating minimal dependence on hole periodicity," Appl. Phys. Lett. 90, 131111 (2007). [CrossRef]
  20. U. C. Fischer, "Submicrometer aperture in a thin metal film as a probe of its microenvironment through enhanced light scattering and fluorescence," J. Opt. Soc. Am. B 3, 1239-1244 (1986). [CrossRef]
  21. H. Rigneault, J. Capoulade, J. Dintinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen and P.-F. Lenne, "Enhancement of Single-Molecule Fluorescence Detection in Subwavelength Apertures," Phys. Rev. Lett. 95, 117401 (2005). [CrossRef] [PubMed]
  22. J. Wenger, B. Cluzel, J. Dintinger, N. Bonod, A.- L. Fehrembach, E. Popov, P.-F. Lenne, T. W. Ebbesen, and H. Rigneault, "Radiative and Nonradiative Photokinetics Alteration Inside a Single Metallic Nanometric Aperture," J. Phys. Chem. C 111, 11469-11474 (2007). [CrossRef]
  23. D. Gerard, J. Wenger, N. Bonod, E. Popov, H. Rigneault, F. Mahdavi, S. Blair, J. Dintinger, and T. W. Ebbesen, "Nanoaperture-enhanced fluorescence: Towards higher detection rates with plasmonic metals," Phys. Rev. B 77, 045413 (2008). [CrossRef]
  24. H. G. Craighead, "Future lab-on-a-chip technologies for interrogating individual molecules," Nature (London) 442, 387-393 (2006). [CrossRef] [PubMed]
  25. J. T. Mannion, and H. G. Craighead, "Nanofluidic Structures for Single Biomolecule Fluorescent Detection," Biopolymers 85, 131-143 (2006). [CrossRef] [PubMed]
  26. C. Genet and T. W. Ebbesen, "Light in tiny holes," Nature (London) 445, 39-46 (2007). [CrossRef] [PubMed]
  27. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead, and W. W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003). [CrossRef] [PubMed]
  28. K. T. Samiee, M. Foquet, L. Guo, E. C. Cox, H. G. Craighead, "Lambda repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides," Biophys. J. 88, 2145-2153 (2005). [CrossRef]
  29. M. Leutenegger, M. Gosch, A. Perentes, P. Hoffmann, O. J. F. Martin, T. Lasser, "Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture," Opt. Express 14, 956-969 (2006). [CrossRef] [PubMed]
  30. J. Wenger, F. Conchonaud, J. Dintinger, L. Wawrezinieck, T. W. Ebbesen, H. Rigneault, D. Marguet, and P. F. Lenne, "Diffusion Analysis within Single Nanometric Apertures Reveals the Ultrafine Cell Membrane Organization," Biophys. J. 92, 913-919 (2007). [CrossRef]
  31. J. Wenger, D. G’erard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture : towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12206-12216 (2006). [CrossRef] [PubMed]
  32. J. Widengren, R. Rigler, and U. Mets, "Triplet-state monitoring by fluorescence correlation spectroscopy," J. Fluoresc. 4, 255-258 (1994). [CrossRef]
  33. J. Widengren, U. Mets, and R. Rigler, "Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study," J. Phys. Chem. 99, 13368-13379 (1995). [CrossRef]
  34. V. Buschmann, K. D. Weston, and M. Sauer, "Spectroscopic Study and Evaluation of Red-Absorbing Fluorescent Dyes," Bioconjugate Chem. 14, 195-204 (2003). [CrossRef]
  35. J. Widengren and P. Schwille, "Characterization of Photoinduced Isomerization and Back-Isomerization of the Cyanine Dye Cy5 by Fluorescence Correlation Spectroscopy," J. Phys. Chem. A 104, 6416-6428 (2000). [CrossRef]
  36. C. Zander, J. Enderlein and R. A. Keller (Eds.), Single-Molecule Detection in Solution - Methods and Applications, (VCH-Wiley, Berlin/New York, 2002). [CrossRef]
  37. F. Mahdavi, Y. Liu, and S. Blair, "Modeling Fluorescence Enhancement from Metallic Nanocavities," Plasmonics 2, 129-142 (2007). [CrossRef]
  38. E. Popov, M. Neviere, J. Wenger, P.-F. Lenne, H. Rigneault, P. Chaumet, N. Bonod, J. Dintinger, and T. W. Ebbesen, "Field enhancement in single subwavelength apertures," J. Opt. Soc. Am. A 23, 2342-2348 (2006). [CrossRef]

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