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A two-dimensional tantalum pentoxide (Ta2O5) photonic crystal (PC) slab with low-background emission was fabricated and a 12-fold enhancement of fluorescence from the organic dyes of perylene diimide adsorbed on the surface of the PCs was observed. The background emissions of the Ta2O5 substrates with and without the PCs after thermal annealing at 600°C with oxygen gas were comparable to that of a well-cleaned cover glass. This is to date the lowest level of background emissions of two-dimensional PCs using materials with a high refractive index (>2). The results reported here provide new insights into the fabrication of the photonic devices that enable highly sensitive fluorescence microscopy or optical detections.
©2011 Optical Society of America
1. Introduction
Photonic crystals (PCs) have been extensively studied over several decades, driven by interest in their properties enabling the control of light propagation using the periodic structures [1,2]. One of the remarkable characteristics of PCs is the ability to control spontaneous radiation [3–5], which leads to low threshold lasers [6,7], highly sensitive biological and medical detections [8], and the control of the photochemical processes of organic molecules [9], among other functions. One particular area of interest has been two-dimensional PC slabs [10,11], which have been used for observing one-photon [12,13] and two-photon [14,15] excited fluorescence enhancement of quantum dots or organic dyes. Although materials with a high refractive index (>2), such as TiO2 [12] or SiN [14], are useful for obtaining an enhancement effect with PCs, they can also have some background emission. The effort to reduce the background emission of the PCs has been dedicated [16], leading to the realization of a highly sensitive fluorescence microscopy and a fluorescence microscopy at single-molecule level [17].
In this study, we fabricate two-dimensional PC slabs using tantalum pentoxide (Ta2O5) for observing an enhancement of fluorescence emission from organic dyes adsorbed on the surface by using fluorescence microscopy. The material of Ta2O5 has a high refractive index (~2) and high transparency in broad wavelength range (0.3-10 μm). While the physical and electrical properties of Ta2O5 have been widely studied in the context of the gate insulator [18,19], studies of optical properties [20] and its applications to PCs [21–23] are still limited. We found that the background emission of the fabricated Ta2O5 PC slabs after thermal annealing with oxygen gas was comparable with that of clean cover glass, and observed the enhancement of the fluorescence emission from the adsorbed organic dyes. The mechanism of the enhancement is discussed with band structures calculated by a three-dimensional plane-wave expansion method.
2. Experimental section
A schematic diagram of a Ta2O5 PC slab with hexagonal lattice is shown in Fig. 1(a) . 50 rows × 50 rows of holes are included in the PC with each lattice constant. A cover glass plate (borosilicate glass) with a refractive index of 1.53 was carefully cleaned with acetone, NaOH solution, and UV ozone cleaner (UV-1, Samco) [24]. Ta2O5 thin film was deposited on the cover glass plate by electron beam (EB) evaporation (Eiko Engineering) with a deposition rate of 1 Å/s. The thickness of the deposited film was 120 nm, as measured by stylus surface profiler (Dektak, ULVAC). To fabricate the PCs, EB lithography (ELS-7700, Elionix) with an EB resist (ZEP520, ZEONREX Electronic Chemicals) of 300 nm thickness and reactive ion etching (RIE) (RIE-10NR, Samco) in a dry process using CHF3 plasma were performed. An average of the etched depths of the Ta2O5 film was 88±3 nm for three times individual processes. After removing the resist layer, the substrate was thermally annealed (GFA-430, Thermo Riko) in O2 at 600°C for 30 min. An annealing temperature of 600°C was chosen because crystallization of Ta2O5 film was reported in annealing above 700°C [25,26]. The refractive index of the Ta2O5 film after the annealing was estimated to be 2.05 by comparison with the numerically calculated transmission spectra of the substrate. 7×10−6 M of N,N'-Bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (PDI) (Santa Cruz Biotechnology) in a toluene solution was spin-coated at 2000 rpm onto the PC substrate. The toluene was spontaneously evaporated during the spin coating. For excitation of the PDI or the substrates, an output of circularly polarized fundamental (976 nm) or second harmonic (488 nm) light of a picosecond (ps) Ti:sapphire laser (Tsunami, Spectra Physics) with 8 MHz repetition was led into an inverted microscope (Eclipse TE2000-U, Nikon) and focused into the upper surface of the PC substrates in air using a 20× (Plan Fluor ELWD 20×, Nikon) or a 100× (LU Plan Apo 100×, Nikon) objective lens. The laser power was measured before entering the inverted microscope. The fluorescence signal was collected by the same objective lens and transmitted through a dichroic mirror and edge filters (BLP01-488R-25, Semrock, and 10SWF-600-B, Newport), and was detected by a photomultiplier tube (PMT) (H7422P-40-MOD, Hamamatsu photonics) [27] which is connected to a time-correlated single-photon counting (TCSPC) module (SPC-630, Becker & Hickl).
Fig. 1 (a) A schematic diagram of a Ta2O5/cover glass photonic crystal (PC) slab. (b) A scanning electron microscope (SEM) image of a PC with a lattice constant of 360 nm fabricated on the Ta2O5/cover glass substrate.
3. Results and discussion
Figure 1(b) shows a scanning electron microscopy (SEM) image of the fabricated Ta2O5 PC with a lattice constant of 360 nm. The surface and the PC part on the film was still amorphous state after the annealing. The polycrystalline structure [26], which could alter the property of the PCs, was not observed. The ratio of the diameter of the air holes (2r) to the lattice constant (a) was measured to be about 0.80 among the PCs with lattice constants of 350-800 nm.
To evaluate the background emission of the Ta2O5 PC slab, a 488 nm second harmonic of ps Ti:sapphire laser was focused on the substrates by a 100 × objective lens, and the emission was measured by using time-correlated single-photon counting (TCSPC). The TCSPC is a widely used method for a highly sensitive time-resolved fluorescence measurement, which counts emitted photons associating with a timing of the exaction laser pulses [17,28,29]. Figure 2(a) and 2(b) shows time-resolved emission signals of a control cover glass plate and the Ta2O5/cover glass substrate, respectively. The control cover glass was carefully cleaned as stated in the previous section to reduce the background emission, whose cleaning processes are used to prepare a substrate for single-molecule spectroscopy [24,30]. Figure 2(c) shows the laser power dependence of emission intensities of the substrates with and without PCs. The background emission of the Ta2O5/cover glass substrate (340 counts/s) was only 13% higher than that of the clean cover glass (300 counts/s) at 2.6 μW excitation. Since the excitation condition of 2.6 μW is the typical experimental condition for single-molecule detection in our experimental setup, these results indicate that the fabricated Ta2O5/cover glass substrate has a very low background emission, applicable to the detection of fluorescent organic dyes at the single-molecule level. To understand the effect on the fabrication of the PC structures, background emissions from the PCs were also investigated (Fig. 2(c)). As can be seen, the intensities of background emission from the PCs with lattice constants of 800 and 600 nm were as low as that of the cover glass, indicating that the material in the vicinity of the holes in the PC slab was also not altered in the fabrication processes. On the other hand, a PC with a lattice constant of 360 nm exhibited a slightly increased background emission, although the count rate was still small (410 counts/s at 2.6 μW excitation). This can be attributed to the enhancement of the emission by the PC, since the Ta2O5 film exhibited the slight emission comparing to the cover glass as described above.
Fig. 2 Time-resolved background emission of (a) a control cover glass and (b) a Ta2O5/cover glass substrate, excited with a ps 488 nm laser of 2.6 μW for 30 s. (c) The laser power dependence of background emission of the control cover glass (crosses), the Ta2O5/cover glass substrate (squares), and PCs with a lattice constant of 360 (circles), 600 (triangles), and 800 (diamonds) nm fabricated on the Ta2O5/cover glass substrate.
In this context, it should be mentioned that the background emission of the Ta2O5/cover glass substrate was greatly decreased by the thermal annealing with O2 gas, although the substrate had a high background emission (>3×103 counts/s) after the RIE with CHF3 plasma. Since the emission of the substrate was also decreased by additional O2 plasma cleaning, some part of the emission can be due to contamination produced by the RIE with CHF3 plasma. On the other hand, an emission from a Ta2O5 substrate without the RIE was decreased by the thermal annealing with O2 gas. Thus, the annealing process with O2 gas have two effects that are burning off the contamination produced by the RIE and reducing the autofluorescence of the Ta2O5 film, which might be due to the oxygen vacancy of the film [31].
We measured the fluorescence decay profiles of the PDI dyes adsorbed on the surface of the PC slabs with lattice constants of 350, 360, 370, 600, and 800 nm. We used the PDI dye because the dye has a high quantum yield and a high photostability and is widely used in fluorescence microscopy [29,30]. A 20× objective lens was employed to obtain the signal from a relatively large spot area with a vertical direction. The coverage factor of the PDI on the surface was estimated to be about 6% from the absorption spectrum of the substrate spin-coated at 2000 rpm with 2.2×10−5 M of PDI solution. Figure 3(a) shows the time profiles of PDI dyes on the PCs with lattice constants of 360, 600, 800 nm, and on the Ta2O5 substrate near (a few μm away from) the PC (hereafter, we refer to as “out of PC”). The strongest intensity was obtained from the PC with a lattice constant of 360 nm. On the other hand, the shortening of the lifetime attributing to Purcell effect was not observed [12] with this lattice constant. Figure 3(b) shows the integrated intensities of the time profiles on the PCs and out of the PCs. The intensities out of the PCs (circles in Fig. 3(b)) were almost the same among the lattice constants, indicating that the PDI dyes were adsorbed uniformly over the substrate. The difference in intensities between the lattice constants of 360 and 370 nm indicates that the resonance of the excitation laser or the fluorescence to the modes of the PCs is significantly affected by the size of the lattice constant. An enhancement factor by PCs in the case of using fluorescence microscopy can be obtained as the ratio of the fluorescence intensity on the PCs to that out of the PCs [13,14], assuming the dye concentrations on the PCs are constant. Figure 3(c) summarizes the enhancement factors among different lattice constants. The enhancement factor at the lattice constant of 360 nm with one-photon excitation was about 12. In Fig. 3(c), the enhancement factor obtained with two-photon excitation (976 nm) in the same manner is also shown. The enhancement factor at a lattice constant of 360 nm was about 3, which is smaller than 12 with one-photon excitation (488 nm). The difference in the enhancement factors between the excitation wavelengths (488 or 976 nm) suggests that both the resonance of the excitation laser and the fluorescence to the modes of the PCs are important for the enhancement [12,14].
Fig. 3 (a) Fluorescence decay profiles of PDI dyes on PCs with a lattice constant of 360 (squares), 600 (triangles), 800 (diamonds) nm and out of PCs (circles) fabricated on the Ta2O5/cover glass substrate, excited with a ps 488 nm laser of 80 nW for 30 s. The inset shows a logarithmic plot of the fluorescence decay profiles. Normalized absorption (red) and fluorescence (blue) spectra, and the PDI structure are shown on the right side of the graph. (b) The lattice constant dependence of the fluorescence intensities of PDI on PC (squares) and out of PC (circles). (c) Enhancement factors of the fluorescence intensities of PDI with one-photon excitation (488 nm) (squares) and two-photon excitation (976 nm) (triangles). The laser power for two-photon excitation was 30 mW.
To discuss the enhancement mechanism, a photonic band structure of Ta2O5 PC slab on cover glass was calculated by a three-dimensional plane-wave expansion method (Fig. 4(a) ) [1,2,32]. A three-dimensional periodic unit cell containing a slab and the surrounding mediums of upper air and a lower cover glass was used in the calculation [32]. Since, in our experimental condition, a 20× objective lens with a relatively small incident angle of 0-27° (N.A. 0.45) is used to inject and collect photons, we consider the coupling of the light with the modes [14] in the region above a line corresponding to the incident angle of 27° (red dashed line), located inside a light cone in which light leaks into external air background [1,2]. Figure 4(b) plots the wavelength of the modes coupling with the incident and emitted light (above the red dashed line) against the lattice constant. As can be seen, the lines of the one-photon excitation laser (488 nm) and the peak wavelengths of the PDI (536 and 575 nm) intersect with the many modes at the lattice constant of 360 nm, indicating that both the excitation and emission wavelengths are resonant with the mode of the PC. On the other hand, the line of the two-photon excitation laser (976 nm) intersects with few modes of the PC at the lattice constants of 360 nm, leading to small enhancement (3-fold). The large enhancement at 360 nm compared with that at 600 and 800 nm with one-photon excitation can be explained by the dominant effect of the lower-order modes in the band diagram.
Fig. 4 (a) A photonic band structure of a Ta2O5 PC slab on cover glass with 2r/a = 0.80 and d = a/3 (circles), where a is a lattice constant, r is a radius of the air hole, and d is a thickness of the slab. The black solid line and the red dashed line respectively represent a light line (ω = ck) in air and a line corresponding to an incident angle of 27° (ω = ck/sinθ), where ω is a frequency, c is the speed of light, k is an in-plane wave vector, and θ is an incident angle. (b) The lattice constant dependence of the wavelength of the modes that couple with the light in the incident angle of 0-27° (see text). Horizontal lines indicate the wavelengths of the excitation lasers (488 and 976 nm) and the fluorescence peaks of PDI (536 and 575 nm). The data point corresponds to the modes above the red dashed line. The high frequency bands above ~1 are not shown.
4. Conclusions
In summary, we fabricated Ta2O5 PCs with very low background emission and obtained an enhancement factor of 12 attributing to the coupling of the excitation laser and the fluorescence to the modes of the PC as for the fluorescence emission from PDI adsorbed on the surface of the PC. The background emission from the Ta2O5 PC slab, even from the PC that exhibited the highest enhancement effect (a = 360 nm), was as low as that from the clean cover glass (<0.5 k counts/s at 2.6 μW excitation). To our knowledge, this is the lowest level of background emission from PC slabs using a material with a high refractive index (>2). Our results are applicable to the enhancement of various weak signals, enabling highly sensitive and efficient biological and optical detections using microscopy, and fluorescence microscopy at single-molecule level.
Acknowledgement
This work was partly supported by Grant-in-Aids for Young Scientists (B) (22750013) and Scientific Research (C) (20510112) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of the Japanese Government.
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