Micro- and nano-patterned fluorescent materials are important for many photonic devices and applications. In this paper, we investigate the impact of three common lithographical techniques, deposition and removal of sacrificial masks, ultraviolet ablation, and focused ion beam milling, on self-assembled fluorophores. We find that different patterning techniques can dramatically change the fluorescence lifetime of the fluorophores and that the degree of modification depends on the patterning techniques.
© 2014 Optical Society of America
Recently, polymer based fluorescent materials have been developed for a large number of applications including imaging , lasing , and chemical / biological sensing [3–5]. For many important applications [3,6–9], micro- and nano-scale patterning of fluorescent materials is essential. A key parameter of the patterned fluorescent materials [10,11] is their quantum yield, which, unfortunately, has not been widely investigated for many commonly used fluorophores and / or lithography techniques. In particular, many recent developments involve polymer based fluorescent materials and colloidal quantum dots (QDs) [3,12]. Numerous patterning techniques have been developed for such fluorescent materials, with examples including methods based on acid-sensitive dyes , photo-bleaching , ultraviolet (UV) ablation , electron beam lithography , and focused ion beam (FIB) milling . More broadly, micro- and nano-patterning of biomolecules have also been demonstrated using electron beam lithography, as recently reviewed in Ref. 18. However, the impact of these micro- and nano-lithography techniques on the fluorescence dynamics of the fluorescent materials has not been thoroughly investigated in the existing literature.
Existing studies on patterning-induced changes of fluorophore properties are based primarily on fluorescence spectrum measurements. For instance, Kocher et al.  found that the patterned photofunctional polymers exhibit photobleaching as well as a blue-shift in emission spectrum. Similarly, UV exposure of various fluorescent polymers can lead to changes in emission spectra . However, certain techniques, such as the soft-molding-based lithography reported in , can be used to pattern functional fluorophores without changing their emission spectra. Yet only a limited number of studies  investigated the impact of lithography (e.g., e-beam lithography) on fluorescence dynamics.
Here we experimentally investigate the impact of three important lithography techniques on the fluorescence behaviors of self-assembled fluorescent materials. Specifically, we study the patterning processes based on: 1) thermal evaporation and chemical removal of masking materials, 2) UV ablation, and 3) FIB milling. The results presented here should be relevant for a large number of applications involving self-assembled fluorescent materials and photonic nanostructures.
2. Patterning based on mask deposition and removal
The process of sacrificial mask deposition and removal is a key component of many micro- and nano-lithography techniques. Here we use the deposition and chemical etching of aluminum masks as an example and investigate its impact on fluorescence dynamics. We consider two types of fluorophores, one is polymer-conjugated Texas Red (TR) dye (Sigma-Aldrich, emission peak at 620 nm) and the other is negatively charged CdSe/ZnS QDs (NN‒LABS, emission peak at 621 nm, 5 nm mean diameter). The TR dye was conjugated to the polycation poly (allylamine hydrochloride) (PAH) using the method in .
To begin lithography, we first cleaned a microscope glass slide  by immersing it in a 1:1:5 solution of NH4OH:H2O2:H2O at 70 °C for 20 min, followed by immersion in a 1:1:6 solution of HCl:H2O2:H2O at 70 °C for additional 5 min. During this process, OH‒ ions were formed on the glass surface to provide a negatively charged layer, which is necessary to initiate the layer-by-layer (LbL) self‒assembly process. Afterwards, we consecutively immersed the negatively charged glass substrate in aqueous solutions of TR-PAH and polyanion poly(styrene sulfonate) (PSS). Due to electrostatic interaction, each deposition step incorporated a layer of TR-PAH or PSS onto the substrate. We repeated this process several times till we deposited three layers of polymers (i.e., TR-PAH/PSS/TR-PAH) onto the glass substrate, as seen in Fig. 1(a).For samples containing QDs, we followed essentially the same approach and deposited a three-layer polymer structure containing PAH/PSS/PAH onto the glass substrate. (The PAH in this case is not conjugated with TR and is therefore transparent.) At the last step, the QDs were adsorbed onto the topmost PAH layer and formed the structure shown in Fig. 1(b). The refractive index of the PAH/PSS film is 1.5 and its thickness is 3 nm per bilayer, as determined by ellipsometry . The atomic force microscope (AFM) images of samples assembled with TR-PAH/PSS/TR-PAH and QDs are shown in Fig. 1(c) and 1(d), respectively.
The photoluminescence of the self-assembled fluorophores was measured using time-correlated single-photon counting (TCSPC), with Picoharp 300 (PicoQuant) and a pulsed 473 nm laser diode (BDL-473-C, Becker and Hickl) as the excitation source. The photoluminescence generated by the samples in Fig. 1(a) and Fig. 1(b) is given in Fig. 1(e) as the “unpatterned” case. The excitation laser was focused onto the sample and formed a Gaussian beam with beam diameter of approximately 3 µm. This paper is not concerned with fluorescence intensity, which depends on a variety of hard to normalize parameters such as QD / dye density. Hence in present study, we only show the normalized photoluminescence data. For ease of comparison, the peak intensity of the “unpatterned” photoluminescence curves was normalized to 0.7 while the “patterned” curves are separately normalized to 1.0.
To investigate the impact of aluminum mask deposition and removal, we first evaporated a 30 nm aluminum layer onto the glass substrate and fluorescent layers using e-beam evaporation (Thermionics Vacuum Products, 60 mA beam current, 5 Å/s evaporation rate.) We select 30 nm for aluminum layer thickness for several reasons. First, thinner films cannot completely block the UV light, which is highly problematic for patterning techniques based on UV ablation. A 30 nm aluminum layer is also sufficient for avoiding the charging issues in FIB milling. On the other hand, choosing a thicker aluminum layer can significantly increase the etching time required for mask removal. After aluminum mask deposition, without patterning the aluminum layer, we immersed the sample in a 1 mM HCl acid for 2 hours at ~45 °C, which was sufficient to completely remove the aluminum deposited over the glass slide. After these steps, we again measure the fluorescence dynamics of the sample covered with TR-PAH and QDs, respectively. The results are shown in Fig. 1(e).
Our experimental results suggest that TR fluorescence is not significantly impacted by aluminum deposition and removal. In fact, the shape of the solid blue line in Fig. 1(e), which shows TR fluorescence after mask removal, is almost identical to the “unpatterned (TR)” result, i.e., the dashed black line. The “tail” portion of TR photoluminescence is somewhat different for the “unpatterned” sample and the sample that has gone through aluminum mask deposition and removal. This difference, however, is likely an artifact caused by the different photon dark count of the two different measurements. In sharp contrast to this behavior, for the glass substrate covered with QDs, after aluminum etching and removal, we can no longer even detect any QD photoluminescence. Either the QDs were removed during HCl etching, or their fluorescence efficiency was significantly reduced by the etching process.
To quantify the fluorescence dynamics shown in Fig. 1, we define average fluorescence lifetime as where I (t) is the experimental measured fluorescence intensity. To further identify any variations in the average lifetime, all experimental results mentioned in this paper were obtained using photoluminescence data measured at 8 distinct locations on the same sample, from which we can determine both the mean and the standard deviation of the lifetime. For the TR results shown in Fig. 1(e), the fluorescence lifetime of the “unpatterned (TR)” sample is 4.03 ± 0.02 ns. In contrast, after aluminum evaporation and removal, the TR lifetime becomes 4.17 ± 0.02 ns. These values confirm that the process of evaporating and etching aluminum has minimal impact on the fluorescence dynamics of TR. For the QD data shown in Fig. 1(e) (i.e., the “unpatterned (QDs)” sample), the lifetime τ was calculated to be 9.8 ± 0.07 ns.
3. Patterning based on UV ablation
In this section, we investigate the impact of UV ablation. The overall fabrication procedure is illustrated in Fig. 2(a).In step 1, we alternately immersed a clean glass substrate in solutions containing TR-PAH and PSS to deposit three layers of TR-PAH/PSS/TR-PAH film. Then, in step 2, we placed a square grid above the sample and used e-beam evaporation to deposit 30 nm aluminum mask over the LbL film. Afterwards, in step 3, we exposed the sample, partially covered by the aluminum mask, to UV ablation. The UV light was provided by a 248 nm Excimer laser, and the exposure time was 5 min, or until the total fluence reached the level of 45 mJ/cm2. Finally, in step 4, the aluminum mask was removed by immersing the sample in a 1 mM HCl aqueous solution for 2 hours at ~45 °C.
Figure 2(b) shows a fluorescence microscope image of the sample after step 2 taken using a Zeiss confocal laser scanning microscope (LSM 510 NLO + VIS). The dark and red regions clearly show sections with and without the aluminum layer, respectively. Similarly, Fig. 2(c) shows a fluorescence image of the sample after step 4, i.e., UV ablation and aluminum removal. Note that the regions protected by the aluminum mask remain fluorescent, whereas regions exposed to UV ablation are no longer fluorescent. These results demonstrate that the 30-nm-thick aluminum mask can effectively protect the TR dye underneath, and that the dose of 45 mJ/cm2 is sufficient to completely remove the self-assembled dye. In Fig. 2(d), we show the photoluminescence of the TR dye measured after step 1 (represented by the dashed black line, i.e., “unpatterned”) and TR fluorescence measured after step 4 (represented by the blue line, i.e., “patterned”). The average TR fluorescence lifetime after the patterning process was 4.18 ± 0.02 ns, and is very similar to the “unpatterned” result of 4.03 ± 0.02 ns. This result confirms that it is possible to use UV ablation to create micron-scale fluorescence patterns without significantly damaging the fluorescent dye or reducing the dye’s quantum yield.
A similar fluorescence patterning process using QDs is outlined in Fig. 3(a). The main difference is that since QDs are either damaged or removed by the process of mask removal, here QDs are self-assembled onto the sample after the patterning process. Obviously, in this case, the QD fluorescence dynamics should not be changed by the patterning process. Specifically, in this method, we used LbL assembly to deposit a PAH/PSS/PAH multilayer structure on a clean glass substrate. The PAH is not conjugated with TR and does not fluoresce. Afterwards, we followed the same UV ablation procedure to pattern a polymeric film capped with a positively charged PAH layer, as illustrated in Fig. 3(a), step 4. In the final step, we immersed the sample in the solution containing negatively charged QDs. Due to the strong electrostatic interaction, almost all QDs were selectively adsorbed onto the regions covered by the PAH/PSS/PAH LbL film, thus generating the patterned structure in step 5.
Figure 3(b) is the fluorescence microscope image of the QD sample fabricated using the aforementioned procedure. The result is clearly consistent with our expectation, and confirms that QDs are predominately adsorbed onto the regions capped with positively charged PAH. Using TCSPC measurements, we find that the QD fluorescence produced by the patterned structure are identical to that of the “unpatterned” sample as shown in Fig. 3(c), and that the average lifetime τ was almost identical, 9.4 ± 0.15 ns. This is to be expected, since QDs were introduced after nanoscale patterning of the substrate.
It should be mentioned that the main reason that we modified the procedure in Fig. 2(a) to the one in Fig. 3(a) is to avoid the step of aluminum mask removal in the presence of QDs. As shown in section 2, the step of HCl etching may either remove the QDs or significantly quench their fluorescence. Thus, in Fig. 3(a), we incorporated QDs after aluminum mask removal to circumvent this problem.
4. FIB-based patterning
The impact of FIB milling is studied in this section. The sample fabrication procedure is schematically shown in Fig. 4(a). In the first step, we deposited a single layer of TR-PAH on a clean glass slide. Afterwards, we assembled 75 bilayers of PAH/PSS above the TR-PAH monolayer in the second step. The PAH used in step 2 was not conjugated with TR and did not generate fluorescence. Then, in step 3, we deposited a layer of 30 nm thick aluminum mask above the LbL film. The aluminum mask serves two purposes. First, it alleviates the charging issue. Second, the presence of the mask may help protecting the fluorophores underneath from unwanted exposure to any potential stray ions. We subsequently used FIB (FEI Helios NanoLab 600, 30 kV acceleration voltage, 9.7 pA beam current, 13 nm ionic beam diameter) to drill an array of nanoscale holes onto the sample in step 4. The diameter of the holes is 300 nm and the lattice spacing is 600 nm. Finally, in step 5, we removed the aluminum mask using the same HCl etching process described in section 2. Figure 4(b) is a SEM image of the patterned structure with aluminum mask, i.e., after step 4. Figure 4(c) is an AFM image of the same structure after step 5, with the aluminum mask removed.
Figure 4(d) shows the TR photoluminescence at different fabrication steps. The fluorescence lifetime measured from underneath (through the glass substrate) after uniform aluminum mask deposition (i.e., after step 3 and before step 4) is shown as the dashed black line. The average lifetime was 2.72 ± 0.02 ns, which is less than the value of TR assembled above glass without the presence of the aluminum film. One possible explanation for the reduced fluorescence lifetime is the presence of the metallic film , which can modify the photonic density of states through the Purcell effect. In fact, if we use 4 bilayers of PSS/PAH (instead of 75 bilayers) to separate the monolayer of TR-PAH from the 30 nm aluminum mask, the average fluorescence lifetime of TR is even smaller, and becomes 1.63 ± 0.07 ns.
The fluorescence decay produced by TR after FIB milling (i.e., after step 4 and before step 5) is shown in Fig. 4(d) as the solid blue line. Our result suggests that the nano-patterned structure could still produce active fluorescent signal, however, the fluorescence lifetime was modified by the patterning process to be 1.84 ± 0.1 ns. Finally, we measured the fluorescence lifetime of the nano-patterned sample after aluminum mask removal (i.e., after step 5). The result is represented in Fig. 4(d) as the dotted red line, which corresponds to an average lifetime of 1.8 ± 0.1 ns. The results in Fig. 4(d) also explain the choice of 75 bilayers of PAH/PSS in our experiments. With sufficiently large distance between the patterned aluminum film and the fluorophores, the Purcell effect due to the presence of the metallic nanostructure is minimized. As a result, removing the nano-patterned aluminum mask has almost no impact on the fluorescence lifetime of the TR dye.
The results in Fig. 4 suggest that FIB milling can be used to generate nanoscale fluorescent patterns. However, the process of FIB milling can substantially reduce the fluorescence lifetime of the self- assembled dye, which may be caused an increase in the non-radiative recombination due to ion damage.
5. Self-assembly of QDs over plasmonic nanostructure
Our results in section 4 clearly suggest that FIB can have significant impact on the fluorescence properties of dyes, and may result in a lower quantum yield. As a result, it is often preferable to incorporate nanomaterials after FIB-based nanolithography. In this section, we consider such an example, where we self-assembled colloidal QDs onto a plasmonic nanostructure, as illustrated in Fig. 5(a).Specifically, as shown in step 1 and 2 in Fig. 5(a), we deposited a layer of gold film (100 nm in thickness) on a glass substrate through e-beam evaporation (Thermionics Vacuum Products, 100 mA beam current, 7 Å/s evaporation rate) (step 1), and used FIB to drill an array of holes onto the gold film (step 2). From the SEM image in Fig. 5(b), the hole diameter is found to be 100 nm and the lattice spacing is 500 nm.
After FIB patterning, we incorporated the QDs onto the plasmonic nanostructure using self-assembly. As shown in step 3 of Fig. 5(a), the patterned gold substrate was immersed in a 1 mM solution of mercaptohexadecanoic acid (MHDA) in ethanol (pH ~2) for 24 hours, followed by ethanol sonication and DI water rinsing. During this step, a monolayer of ω-COOH thiol was adsorbed onto the gold film to form a negatively charged layer. Afterwards, in step 4, the sample was immersed consecutively in aqueous solutions of PAH and PSS till we self-assembled the desired number of layers capped with PAH. Finally, in step 5, we immersed the sample in the solution of the colloidal QDs for self-assembly. In the present study, the QDs and the plasmonic structure were separated by four bilayers of PAH/PSS plus a single additional layer of PAH above the thiol. Figure 5(c) shows a fluorescent image of the final structure, with QDs assembled over the plasmonic substrate. (The fluorescence image was obtained using Zeiss confocal laser scanning microscope (LSM 510 NLO + VIS).) We observed a bright spot that corresponds to the nano-patterned region in Fig. 5(b). Somewhat surprisingly, the rest of the structure, which is unpatterned but does contain self-assembled QDs, produces significantly less fluorescence.
The fluorescence lifetime of the assembled QDs is measured using the same TCSPC system. Figure 5(d) shows the fluorescence decay produced by the unpatterned section (the dashed black line) and patterned section (the solid blue line), respectively. The average lifetime τ is found to be 4.1 ± 0.08 ns for the unpatterned section and 3.98 ± 0.09 ns for the patterned section, respectively.
The results in Fig. 5(d) suggest that the patterned plasmonic substrate does not significantly modify the fluorescence lifetime of the QDs, but can produce much stronger fluorescence intensity. The enhanced fluorescence intensity in the patterned region can perhaps be explained by the scattering of the nanoholes, which can convert the non-radiative plasmonic excitations into radiative signals. A full investigation of this phenomenon, however, requires much more detailed investigation of plasmonic excitations in the nano-patterned structure and is outside the scope of this paper.
6. Discussion and conclusion
We investigate the impact of several commonly used lithography techniques on the fluorescence properties of self-assembled fluorophores. We find that the process of aluminum mask deposition and removal does not significantly change the fluorescence lifetime of TR dye. One the other hand, QDs is more “finicky”, and can be easily damaged or removed by the process of aluminum mask removal. To circumvent this problem, for fluorescent structures containing QDs, one can deposit / incorporate QDs during the last stage of sample fabrication.
In this paper, we study two types of micro- and nano-patterned samples: micro-patterned samples produced through UV ablation, and nano-patterned samples fabricated using FIB milling.
For the micro-patterned samples, we demonstrate that a proper combination of UV ablation and self-assembly can be used to generate micron-scale patterns of fluorescent dyes and QDs without any significant changes on their fluorescence dynamics. In particular, for the dye based samples, we can incorporate fluorescent dyes before sample lithography. However, for the QD based samples, due to the problem of mask removal, QDs are deposited after the patterning of the transparent polymer coating. Due to the electrostatic interaction between the QDs and the polymers, the QDs are selectively deposited onto the regions covered with the polymers. For the nano-patterned samples, we find that the process of FIB milling can significantly change the fluorescence dynamics of TR dyes. Finally, we incorporate QDs onto a nano-patterned Au structures. We find that the sample with patterned gold substrate and the one with flat gold substrate have similar QD fluorescence dynamics. However, the patterned structure can significantly enhance the fluorescent intensity produced by the QDs.
We would like to thank the National Science Foundation (grants ECCS-0644488 and 1128587) and the VT-MENA program for generous support.
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