2. 16. L. Li, E. H. Chen, J. Zheng, S. L. Mouradian, F. Dolde, T. Schröder, S. Karaveli, M. L. Markham, D. J.
Twitchen, and D. Englund, “Efficient photon collection from a nitrogen vacancy center in a circular bullseye
grating,” Nano Lett. 15(3), 1493–1497 (2015).
17. H. Shen, G. Lu, T. Zhang, J. Liu, and Q. Gong, “Enhanced single-molecule spontaneous emission in an
optimized nanoantenna with plasmonic gratings,” Plasmonics 8(2), 869–875 (2013).
18. H. Shen, G. Lu, T. Zhang, J. Liu, Y. He, Y. Wang, and Q. Gong, “Molecule fluorescence modified by a slit-
based nanoantenna with dual gratings,” J. Opt. Soc. Am. B 30(9), 2420 (2013).
19. C. Sönnichsen, S. Geier, N. E. Hecker, G. von Plessen, J. Feldmann, H. Ditlbacher, B. Lamprecht, J. R. Krenn,
F. R. Aussenegg, V. Z. H. Chan, J. P. Spatz, and M. Möller, “Spectroscopy of single metallic nanoparticles using
total internal reflection microscopy,” Appl. Phys. Lett. 77(19), 2949 (2000).
20. D. A. Woods and C. D. Bain, “Total internal reflection spectroscopy for studying soft matter,” Soft Matter 10(8),
1071–1096 (2014).
21. G. Lu, W. Li, T. Zhang, S. Yue, J. Liu, L. Hou, Z. Li, and Q. Gong, “Plasmonic-enhanced molecular
fluorescence within isolated bowtie nano-apertures,” ACS Nano 6(2), 1438–1448 (2012).
22. M. A. Lieb, J. M. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern
imaging,” J. Opt. Soc. Am. B 21(6), 1210 (2004).
23. A. L. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces
investigated with back focal plane imaging,” J. Biomed. Opt. 10(5), 054007 (2005).
24. R. Y. Chou, G. Lu, H. Shen, Y. He, Y. Cheng, P. Perriat, M. Martini, O. Tillement, and Q. Gong, “A hybrid
nanoantenna for highly enhanced directional spontaneous emission,” J. Appl. Phys. 115(24), 244310 (2014).
25. 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(3), 2276–2287 (2008).
1. Introduction
Molecular fluorescence detection has various applications in many fields such as biosensors,
early diagnosis, bioimaging, single photon source and so on [1,2]. However, the low signal to
noise ratio (SNR) limits the detection efficiency of single molecule fluorescence. Thus, how
to control the photoluminescence process, which can be divided into excitation and emission
processes, becomes a main topic in this field. As we know, metallic nanostructures have
novel characteristics, i.e. surface plasmon (SP) and localized surface plasmon (LSP) [3,4],
which can be exploited to modify the photoluminescence process strongly [5–7]. For instance,
metallic nano-apertures are able to confine electromagnetic field within the aperture due to
LSP effect, and the resulted high intensity field can enhance the excitation efficiency. Also,
the nano-apertures have an extra advantage of suppressing background [8–10]. In addition,
plasmonic gratings have attracted many attentions due to their unique SP characteristic. The
plasmonic gratings cannot only enhance specific SP mode but also modify its propagation
direction. For example, plasmonic gratings are able to modify the light transmission from
single nano-aperture [11]. Moreover, the hybrid nanostructure of plasmonic gratings and
nano-aperture have been extensively investigated for single molecule fluorescence
enhancement [12–16]. When the gratings coupling with the nano-aperture, it cannot only
enhance the near-field excitation rate but also it can modify the emission rate and the far-field
radiation pattern of the molecular fluorescence. It is also found that the resonance wavelength
is dependent on their geometry parameters, such as the groove depth and grating period,
which provide a way to optimize the optical response corresponding to specific fluorescence
molecule [17,18]. While conventional plasmonic gratings response effectively only in a
narrow spectral band. Therefore, there is a tradeoff between the excitation and emission
processes when the Stokes shift of the photoluminescence process is relatively large.
Although asymmetric dual-face grating antenna was proposed in theory to control the local
excitation enhancement, the collection efficiency, and the quantum efficiency separately, the
goals to optimize the fluorescence enhancement still remain an open challenge in experiment.
In this study, we demonstrated that the nano-apertures associated with stair-gratings have
high surface enhancement factor and better beaming effect in comparison to the conventional
ones. In contrast to the conventional gratings, we propose to excavate a rectangle part of
corrugations and make the cross profile likes a stair-grating. The schematic of stair-gratings is
shown in Fig. 1 as a hybrid of two gratings with different period or depth. Thus, a new
periodic parameter is introduced into the plasmonic grating, which could increase the optical
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19568
3. response both at the excitation and emission bands simultaneously. In experiment, we used
fluorescence correlation spectroscopy (FCS) to analyze the fluorescence trace and the
fluorescence count rate per molecule. The nano-apertures with stair-gratings truly presented
higher enhancement effect. In addition, the emission angular patterns were measured with the
back focal plane (BFP) imaging method, presenting a narrower directionality for the stair-
gratings. By employing finite-difference time-domain (FDTD), the directional emission
patterns and near-field enhancement were calculated. The numerical simulations are in good
agreement with the experiments. The proposed stair gratings provide a flexible way to control
the enhancement and beaming effect of the molecule fluorescence.
Fig. 1. Schematic of proposed stair-gratings. We excavate a rectangle part of the corrugations
and make it like a stair. There are two new geometry parameters which can use to tune the
optical response. (b) and (c) SEM cross profile of the common grating and stair-grating
separately.
2. Experimental methods
In the experiment, optical measurement methods are set up on an integrated microscopy
system (NTEGRA Spectra, NT-MDT). The schematic of optical setup is shown in Fig. 2(a).
We can measure white light dark-field scattering [19,20], photoluminescence(PL), FCS
[12,21], and fluorescence radiation patterns [22,23] of the same single nano-aperture in situ.
In all measurements, we used an oil-immersion objective lens (N.A. 1.49, 60 × , TIRF,
Olympus). And the angular patterns of the fluorescence emission were obtained with the back
focal plane (BFP) imaging method. A CW laser at wavelength of 632.8 nm was used as the
excitation light with excitation power of ~60 μW. On the other hand, we used Alexa 647 in
the solution with concentration of ~1μM. To prevent from molecular adsorption due to the
local charges, we used phosphate aqueous buffered solution. To prepare the metallic
nanostructures, there are four steps as shown in Fig. 2(c): (1) Deposit Au thin film on the
cover glass by using magnetic sputtering; (2) Use focus ion beam (FIB) to penetrate through
the Au film and etch into the glass to fabricate the corrugations of the gratings; (3) Deposit
Au film again onto the samples in order to fill the corrugations; (4) Fabricate the central nano-
aperture with FIB milling. By using the Pt deposition which is integrated in the FIB system,
we can easily identify the fabricated nanostructure under the SEM. The SEM images of the
common and stair gratings are shown in Figs. 1(b) and 1(c) separately. We find that the
corrugations of the stair-gratings and the common gratings can be fabricated well as expected.
In our experiments: the corrugation groove depth d = 200 nm, width a = 220 nm, the height of
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19569
4. the excavated part d2 = 100 nm, the width of the excavated part a2 = 110, the central aperture
diameter (same with bare aperture) D = 250 nm, there are 5 grooves for both the stair and
common gratings, and Au film thickness H = 300 nm.
Fig. 2. (a) Schematic of optical experiment setup. (b) Optical confocal scanning image of a
sample containing bare apertures, nano-apertures surrounded with common and stair grating.
(c) Fabrication procedure of the nano-apertures with stair-gratings.
3. Results and discussion
First of all, we use the white light dark-field scattering method to characterize the response of
the nanostructures. The results are shown in Fig. 3(a). As we can see, in the range from 625
nm to 675 nm, both the stair-gratings (red line) and the common gratings (blue line) present a
broad resonance. Obviously, the stair-gratings have higher scattering intensity which implies
it has stronger optical response when comparing to the common gratings. Correspondingly,
the Alexa 647 dye molecules used in the present experiment also emits in this range. Thus,
spectral response of the nanostructures covers not only the excitation wavelength of 632.8 nm
but also the emission spectral range of 640 ~700 nm. Figure 3(b) shows the fluorescence
spectra of the molecules being confined within the nano-apertures. The results from three
kinds of nanostructures are compared: bare nano-apertures, nano-apertures with common
gratings, and nano-apertures with stair-gratings. As shown in Fig. 3(b), the nano-apertures
with gratings modify the fluorescent spectral shape slightly when comparing to the
fluorescence spectrum obtained from the dye molecules in free solution (data not shown). At
wavelength ~700 nm, there is a “shoulder” for both the common and stair gratings. This
spectral variation can be due to the strong interaction between the dye molecules and the
nanostructures [10]. The PL spectrum of the dye molecules in free solution is also plotted for
comparison.
In order to obtain normalized fluorescence count rate per molecule, it is necessary to
obtain the average number of the dye molecules in the nano-apertures. In the following, we
measured the fluorescence intensity from single nano-apertures with two avalanche
photodiodes (SPCM-AQRH-16-FC, PerkinElmer) and obtained the FCS curves through
cross-correlation to suppress after-pulsing (correlator.com, US). The results are shown in Fig.
4(a). The FCS curves of three kinds of nanostructures have little difference, which means that
the average number of the molecules is almost the same because the different nanostructures
actually have the same nano-aperture size. Nevertheless, we notice that the result of the bare
aperture has a slightly higher G(0) which probably due to less background noise than the
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19570
5. others. According to three dimensional Brownian diffusion model, we have the formula:
( ) ( )
12
1/221
1 1 1 1 1 /T d
bT d
B
G n exp s
N F
τ τ
τ τ τ
τ τ
−
−
= + − + − + +
[8,10,21], where N is the
total number of molecules, F the total signal, B the background noise, nT the amplitude of the
dark state population, bT the dark state blinking time, τd the mean diffusion time, and s the
ratio of transversal to axial dimensions of the analysis volume. We used this formula to fit the
FCS curves and calculate the average number of molecules. After fitting the curves, we
obtained the fluorescence count rate per molecule. As shown Fig. 4(c), we provide the
averaged count rate and standard deviation in different structures, summarized from three
stair-gratings, four common gratings and four bare apertures. In average, in comparing to the
bare aperture, the stair-gratings can reach 2-fold fluorescence enhancement factor. And the
stair-gratings perform better than common gratings. The stair-gratings have the highest count
rate, i.e. the stair-gratings can still promote 20% of the count rate than the common ones.
Both the count rate of the common gratings and the stair-gratings are much higher than that of
the bare nano-apertures. It implies that the stair-gratings are able to significantly enhance the
fluorescence.
Fig. 3. (a) Scattering spectra of nano-aperture with stair and common gratings. (b)
Fluorescence spectra of the molecules within different nanostructures: stair-gratings, common
gratings and bare aperture. PL spectrum in free solution is also plotted for comparison.
Fig. 4. (a) Fluorescence correlation spectroscopy curves of three different nanostructures. (b)
Normalized representative fluorescence intensity trace, and (c) Normalized fluorescence count
rate per molecule for different structures.
Additionally, it is necessary to discuss the molecular far-field radiation pattern modified
by the different nanostructures, since it is another important factor to determine the collection
efficiency during the surface enhanced fluorescence. Figure 5 shows the fluorescence far-field
radiation patterns with the BFP imaging method, and all images with the same color bar.
Figure 5(a) shows the results of the bare apertures and the distribution of the pattern is
homogeneous. It implies that the molecular radiation within the bare aperture spread in a
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19571
6. broad solid angle. The patterns of the common gratings are shown in Fig. 5(b). As we can see,
the fluorescence signals concentrate more in the center, which means that the common
gratings are able to boost the collection efficiency. Furthermore, as shown in Fig. 5(c), we
find that the fluorescence signals are highly confined in the center when comparing to the
former two nanostructures. These results imply that the stair-gratings can confine the
molecular radiation in a narrower solid angles. We notice that the experimental results above
are based on the use of the high N.A. objective (N.A. = 1.49) which can collect the
fluorescence signal very efficiently. Hence, we can obtain a higher enhancement factor if we
use an air objective to replace the oil immersion objective.
Fig. 5. Molecular radiation patterns from different structures (a) Bare nano-aperture (b) Nano-
aperture with common gratings (c) Nano-aperture with stair-gratings.
Fig. 6. Simulated far-field radiation patterns at different wavelengths for (a) Bare nano-
aperture (b) Nano-aperture with common gratings (c) Nano-aperture with stair-gratings, and
near-field intensity enhancement indicated in each top-right corner correspondingly.
Furthermore, we employed the FDTD method to simulate the phenomena qualitatively
[24]. In our simulations: the corrugations height d = 200 nm, width a = 220 nm, the height of
the excavated part d2 = 100 nm, the width of the excavated part a2 = 110, the central aperture
diameter (same with bare aperture) D = 250 nm, Au film thickness H = 300 nm. For the
wavelength from 633 nm to 672 nm, we calculated the near-field enhancement factor within
the nano-apertures and the far-field radiation patterns. It should be note that we only present
the results for the dipole is parallel to the Au film, because such orientation is dominated over
the perpendicular ones. For instance, the power radiative of parallel dipole is about two order
of magnitudes higher than the perpendicular one. As shown in Fig. 6(b), the common gratings
perform well at the wavelength of 633 nm, but the efficiency of the common gratings
decrease for longer wavelength significantly. It is due to the width of the spectral response is
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19572
7. narrower. In contrast, as shown in Fig. 6(c), the stair-gratings perform well at the wavelength
of 633 nm. And the performance of the stair-gratings at wavelength of 650 ~670 nm are still
good enough for good beaming effect although the excitation enhancement factor is lower
slightly. These simulation results are in good agreement with the experiments, i.e. the stairs-
gratings can confine the radiation better than the common gratings. It should be noted that the
groove depth in the present study is pretty deep according previous studies [12,25]. It is still
necessary to optimize the parameters of the stair grating, e.g. groove depth and geometry of
excavated part so on, for better performance. Nevertheless, based on current experimental and
numerical simulations, it is reliable to conclude that the stair grating can work better rather
than the common grating.
4. Conclusions
In conclusion, we design a new type of gratings called as stair-gratings and combine them
with nano-aperture for surface enhanced fluorescence detection. In comparison with the
conventional ones, we find that the detected fluorescent intensity by the stair-gratings is
higher than the common grating. And narrower directionality by the stair-gratings would
enable the detection of molecular fluorescence with low N.A. objective. All these factors
allow a higher SNR and higher detection efficiency of single molecule fluorescence. We also
employed the FDTD method to simulate the near-field enhancement and far-field radiation
patterns. The simulation results are in good agreement with the experimental results. Our
research contributes to understanding to the plasmonic gratings for optimizing the surface
enhancement process of photoluminescence process.
Funding
This work was supported by the National Key Basic Research Program of China (grant no.
2013CB328703) and the National Natural Science Foundation of China (NSFC) (grant nos.
61422502, 11374026, 61521004, 11527901)
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19573
![16. L. Li, E. H. Chen, J. Zheng, S. L. Mouradian, F. Dolde, T. Schröder, S. Karaveli, M. L. Markham, D. J.
Twitchen, and D. Englund, “Efficient photon collection from a nitrogen vacancy center in a circular bullseye
grating,” Nano Lett. 15(3), 1493–1497 (2015).
17. H. Shen, G. Lu, T. Zhang, J. Liu, and Q. Gong, “Enhanced single-molecule spontaneous emission in an
optimized nanoantenna with plasmonic gratings,” Plasmonics 8(2), 869–875 (2013).
18. H. Shen, G. Lu, T. Zhang, J. Liu, Y. He, Y. Wang, and Q. Gong, “Molecule fluorescence modified by a slit-
based nanoantenna with dual gratings,” J. Opt. Soc. Am. B 30(9), 2420 (2013).
19. C. Sönnichsen, S. Geier, N. E. Hecker, G. von Plessen, J. Feldmann, H. Ditlbacher, B. Lamprecht, J. R. Krenn,
F. R. Aussenegg, V. Z. H. Chan, J. P. Spatz, and M. Möller, “Spectroscopy of single metallic nanoparticles using
total internal reflection microscopy,” Appl. Phys. Lett. 77(19), 2949 (2000).
20. D. A. Woods and C. D. Bain, “Total internal reflection spectroscopy for studying soft matter,” Soft Matter 10(8),
1071–1096 (2014).
21. G. Lu, W. Li, T. Zhang, S. Yue, J. Liu, L. Hou, Z. Li, and Q. Gong, “Plasmonic-enhanced molecular
fluorescence within isolated bowtie nano-apertures,” ACS Nano 6(2), 1438–1448 (2012).
22. M. A. Lieb, J. M. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern
imaging,” J. Opt. Soc. Am. B 21(6), 1210 (2004).
23. A. L. Mattheyses and D. Axelrod, “Fluorescence emission patterns near glass and metal-coated surfaces
investigated with back focal plane imaging,” J. Biomed. Opt. 10(5), 054007 (2005).
24. R. Y. Chou, G. Lu, H. Shen, Y. He, Y. Cheng, P. Perriat, M. Martini, O. Tillement, and Q. Gong, “A hybrid
nanoantenna for highly enhanced directional spontaneous emission,” J. Appl. Phys. 115(24), 244310 (2014).
25. 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(3), 2276–2287 (2008).
1. Introduction
Molecular fluorescence detection has various applications in many fields such as biosensors,
early diagnosis, bioimaging, single photon source and so on [1,2]. However, the low signal to
noise ratio (SNR) limits the detection efficiency of single molecule fluorescence. Thus, how
to control the photoluminescence process, which can be divided into excitation and emission
processes, becomes a main topic in this field. As we know, metallic nanostructures have
novel characteristics, i.e. surface plasmon (SP) and localized surface plasmon (LSP) [3,4],
which can be exploited to modify the photoluminescence process strongly [5–7]. For instance,
metallic nano-apertures are able to confine electromagnetic field within the aperture due to
LSP effect, and the resulted high intensity field can enhance the excitation efficiency. Also,
the nano-apertures have an extra advantage of suppressing background [8–10]. In addition,
plasmonic gratings have attracted many attentions due to their unique SP characteristic. The
plasmonic gratings cannot only enhance specific SP mode but also modify its propagation
direction. For example, plasmonic gratings are able to modify the light transmission from
single nano-aperture [11]. Moreover, the hybrid nanostructure of plasmonic gratings and
nano-aperture have been extensively investigated for single molecule fluorescence
enhancement [12–16]. When the gratings coupling with the nano-aperture, it cannot only
enhance the near-field excitation rate but also it can modify the emission rate and the far-field
radiation pattern of the molecular fluorescence. It is also found that the resonance wavelength
is dependent on their geometry parameters, such as the groove depth and grating period,
which provide a way to optimize the optical response corresponding to specific fluorescence
molecule [17,18]. While conventional plasmonic gratings response effectively only in a
narrow spectral band. Therefore, there is a tradeoff between the excitation and emission
processes when the Stokes shift of the photoluminescence process is relatively large.
Although asymmetric dual-face grating antenna was proposed in theory to control the local
excitation enhancement, the collection efficiency, and the quantum efficiency separately, the
goals to optimize the fluorescence enhancement still remain an open challenge in experiment.
In this study, we demonstrated that the nano-apertures associated with stair-gratings have
high surface enhancement factor and better beaming effect in comparison to the conventional
ones. In contrast to the conventional gratings, we propose to excavate a rectangle part of
corrugations and make the cross profile likes a stair-grating. The schematic of stair-gratings is
shown in Fig. 1 as a hybrid of two gratings with different period or depth. Thus, a new
periodic parameter is introduced into the plasmonic grating, which could increase the optical
Vol. 24, No. 17 | 22 Aug 2016 | OPTICS EXPRESS 19568](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)