1. Introduction

Photonic nanostructures are receiving a considerable interest to enhance and control the luminescence properties of a single quantum emitter [1–4]. In the search of deterministic and highly reproducible nanoantennas, much attention has been devoted to subwavelength apertures milled in metallic films [5]. Adding a periodic circular grating (also known as the Bull’s eye structure) around the central aperture enables further control on the light-matter interaction, to improve the transmission efficiency and directionality [6, 7], enhance the non-linear conversion efficiency [8], or realize a photodiode with high-speed response [9]. An exhaustive exploration of the design parameters defining the corrugated nanoaperture is now at hand for the optical transmission process [10]. For the molecular fluorescence process, it has been demonstrated very recently that an optimized nanoaperture surrounded by 5 circular corrugations can significantly enhance the fluorescence count rates per molecule and control the emission directivity [11]. Many studies still deserve to be done to understand how the many design parameters for corrugated nanoapertures influence the fluorescence enhancement process.

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

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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] and in the supporting information of [11], 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.

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To distinguish the relative contributions of the excitation and emission gains in the overall fluorescence enhancement factor, we have developed a specific procedure [12]. Briefly, the fluorescence CRM is measured by FCS while increasing the excitation power I_e. This set of data is fitted by the standard expression of the fluorescence count rate per molecule: CRM = AI_e/(1 + I_e/I_s), where A is a constant proportional to the molecular absorption cross-section, emission rate and setup collection efficiency and I_s is the saturation power. In the saturation regime (I_e ≫ I_s), CRM = AI_s 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 AI_s of the fitting parameters, is equivalent to the emission gain η_em brought by the antenna. This emission gain η_em is expressed as the product of the radiative rate enhancement η_rad times the collection efficiency enhancement η_κ: η_em = η_radη_κ. 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 I_s and the CRM asymptotic limit AI_s.

In the weak excitation regime (I_e ≪ I_s), the fluorescence enhancement equals η_F = CRM_aperture/CRM_solution = A_aperture/A_solution. This can be specified as η_F = η_κη_ϕη_exc, [1] where η_κ is the collection efficiency enhancement, η_exc is the excitation intensity enhancement and η_ϕ = η_rad/η_tot is the quantum efficiency enhancement (ratio of radiative rate enhancement η_rad to the modification of the total fluorescence decay rate η_tot). The alteration in the total fluorescence decay rate η_tot is determined separately by fluorescence lifetime measurements using the standard Time-Correlated Single Photon Counting (TCSPC) method, which is detailed in Ref. [12]. To quantify the excitation intensity gain η_exc, we rewrite the fluorescence enhancement as η_F = η_excη_em/η_tot to make apparent the emission gain η_em. The separate knowledge of η_F, η_em and η_tot finally quantifies the excitation intensity gain η_exc. This procedure separates the excitation, emission, and decay rate contributions. Please note that the emission gain η_em contains a term proportional to the setup collection efficiency. Therefore, the ratio η_em/η_tot = η_κη_ϕ is greater than the quantum yield enhancement of the fluorophore [11]. More details about this procedure can be found in reference [12].

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].

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, 11]. 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

We start our investigations by determining the CRM while increasing the excitation power from 0.1 to 1 mW. Results as measured by FCS are presented in Fig. 2(b), together with the numerical fits according to the model CRM = AI_e/(1 + I_e/I_s). The fit parameters A, I_s are summarized in Fig. 2(c). Large CRM values are readily obtained with corrugated nanoapertures even in the case of low excitation powers below 100 μW, hereby demonstrating single molecule sensitivity with a simple low NA lens. It is also apparent that corrugated apertures provide much higher fluorescence count rates than non-corrugated apertures, with a growing CRM as the number of corrugations is increased. This is a clear indication of the influence of the plasmonic corrugations surrounding the apertures. Please note that these results are highly reproducible, with a relative uncertainty of 10% on the CRM corresponding to the minor variations from a structure to the next and to the repeatability of measurements on our setup.

To quantify the influence of the circular corrugations, we compute the fluorescence enhancement in the low excitation limit η_F = CRM_aperture/CRM_solution = A_aperture/A_solution (Fig. 2(d)). We observe a growing behavior of the enhancement factors as the number of corrugations is increased from 0 (η_F = 14.5) up to 5 (η_F = 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 (η_F = 51.5) as compared to a bare aperture. This constitutes the first experimental observation of the effect numerically predicted in [13], and stands in good agreement with the values inferred in this reference.

The next step determines the respective contributions of excitation and emission gains in the global fluorescence enhancement, following the procedure described in Section 2.2. Figure 3 summarizes our results for the different nanoapertures. Both excitation and emission gains are higher in the presence of corrugations than for a non-corrugated aperture. The concentric corrugations clearly improve the antenna capability of the central aperture to reversibly couple the far field radiation to near-field energy. The excitation gain gradually increases with the number of corrugations, indicating a stronger excitation energy concentration in the central aperture. The emission gain appears almost equivalent for the different numbers of corrugations. This indicates that a single corrugation already enables strong concentration of the fluorescence emission towards the detectors, as we will discuss below. This set of values forms the first complete characterization quantifying the influence of the number of corrugations on the excitation and emission gains for molecular fluorescence enhancement.

 

Fig. 3 Excitation η_exc, emission η_em and total decay rate η_tot gains contributing to the global fluorescence enhancement η_F = η_excη_em/η_tot.

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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).

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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]. These results and the many opportunities for further optimization [10, 13] open promising routes for ultimate control of the emission from a single quantum emitter [1].