1. Introduction

Polymeric microstructures have been envisioned as potential candidates for the next generation of technological materials [1–3]. In recent years, various methods have been proposed aiming at producing polymer-based microstructures to manufacture microdevices. Among them, two-photon polymerization (2PP) has emerged as a promising technique for fabricating microdevices due to the benefits it provides. Although the first report of polymerization initiated via two-photon absorption dates back to 1965 [4], it gained importance when it was proposed as a technology for the microfabrication of devices [5]. Because of its spatial selectivity, 2PP enables the fabrication of complex three-dimensional structures without geometrical limitation, such as structures with moving parts.

In the last few years, the 2PP fabrication of microstructures containing compounds of interest has been demonstrated [6–8], with interesting prospects for the development of applications [8,9]. These structures can be specially designed to meet the desired application needs. For instance, concerning biological applications, microstructures doped with organic compounds have been used for tissue engineering and drug delivery studies [10,11].

Despite recent advances in techniques to fabricate doped microstructures, such approaches have usually been limited to the incorporation of single dopants. Therefore, literature reporting the use of 2PP to produce microstructures doped with two or more compounds is still scarce. Microstructures fabricated by 2PP, consisting of lines with distinct dyes, have been recently reported [12]. Microstructures containing multiple dopants are interesting to the development of special technologies in different fields of science, from optics to biology. For instance, one could use multiple doped 2PP microstructures for producing structures with RGB standard fluorescence or, in the case of tissue engineering, use distinct growth factor with specific action on cells.

In this paper we fabricate three-dimensional microstructures doped with two or more fluorescent dyes (Stilbene 420, Disodium Fluorescein and Rhodamine B), using 2PP. Such microstructures, containing different dyes at specific sites, exhibit a broad fluorescence spectrum, from approximately 350 nm up to 700 nm, whose relative intensity can be altered by selecting the excitation wavelength or the spatial position where excitation is carried out. Therefore, such results point out for an interesting approach to design multi-doped microstructures, presenting tunable and broad fluorescence spectrum, that can be use for several optical applications.

2. Experimental

In the 2PP technique, a femtosecond laser beam is focused through a microscope objective in the volume of a polymeric resin containing a photoinitiator, a compound which is responsible to start the polymerization. In general, the intensity of femtosecond pulses is high enough to induce nonlinear absorption (two-photon absorption) at the focal volume. The quadratic dependence on the intensity, exhibited by the two-photon absorption process, allows spatial confinement of the excitation to the focal volume and, consequently, of the polymerization, providing high spatial resolution to the 2PP technique. We used 100-fs pulses from a Ti:sapphire laser oscillator operating at 780 nm. The structures were fabricated using an average laser power of approximately 40 mW and a 0.25-NA objective that focuses the laser beam into the resin. Our system allows real time visualization of microfabrication, using a red LED as illumination source and a CCD camera coupled to the experimental setup. The sample was positioned in the axial z-direction using a motorized stage, and the laser beam was scanned in the resin x-y-directions with a set of galvanometric mirrors. Further details about the experimental system employed for the fabrication of microstructures via two-photon polymerization can be obtained elsewhere [13].

The basic polymeric resin employed in this work contains two three-acrylate monomers; while tris(2-hydroxyethyl)isocyanurate triacrylate (50 wt.%) gives hardness to the structure, the ethoxylated(6) trimethyl-lolpropane triacrylate (50 wt.%) reduces the shrinkage tensions upon polymerization [14]. The monomers, together with the photoinitiator (Lucirin TPO-L) [14], are mixed for 1 hour to obtain a homogeneous solution. In this work, we used Stilbene 420, Disodium Fluorescein and Rhodamine B as dopants to the microstructures. The dyes are commercially available in the form of powders. To add the dyes to the host resin, initially we dissolved each of the dyes in separate ethanol solutions and added the solutions to the monomers. When the dopants are added to the resin, the solutions are left to mix for 1 hour and, afterwards, the mixtures are left to rest for 24 hours for solvent evaporation.

The fabrication of microstructures containing two or more dopants is carried out in stages. Initially we put a drop of resin doped with, for example, Rhodamine B, in a glass slide, where the resin remains allocated between two spacers and is enclosed by a cover slip. After fabricating a portion of the microstructures by 2PP, the sample is immersed in ethanol for approximately 15 minutes to remove the unpolymerized resin. To make the second part of the microstructure, doped with another dye (e.g., Disodium Fluorescein), we put a drop of this second doped resin on the same substrate already containing the first portion of the microfabricated structured. After the second microfabrication, again we repeat the washing procedure in ethanol. This method does not limit the shape or the number of microstructures which may be produced. The fabricated microstructures were characterized by scanning electron and confocal fluorescence microscopies. The confocal microscopy images of the microstructures were obtained using 350 nm light (40 × objective). The emission spectra of microstructures were obtained by a spectrometer coupled to an optical microscope. As the excitation source for fluorescence we used a He-Cd laser operating at 325nm.

3. Results and discussion

In Fig. 1 it is presented the fluorescence spectra of single doped 2PP microstructures, with Stilbene 420 (blue curve), Disodium fluorescein (green curve) and Rhodamine B (red curve), respectively. From Fig. 1 we observe that the emission peak of Stilbene 420, Disodium Fluorescein and Rhodamine B are, respectively, 420 nm, 535 nm and 580 nm. Such values are in good agreement with the ones reported in the literature for these dyes [15–17], indicating that the dyes were not degraded during the microfabrication and are retained in the microstructures after the washing process, which is desirable if this method were to be used for the fabrication of optical devices.

 

Fig. 1 Emission spectrum of microstructures doped with Stilbene 420 (blue), Disodium Fluorescein (green) and Rhodamine B (red).

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The scanning electron microscope (SEM) image (top view) of a double doped microstructure (Disodium Fluorescein and Rhodamine B) is presented in Fig. 2(a) . Such image reveals that the microstructure exhibits good integrity and definition, indicating that the presence of the dyes does not greatly affect the 2PP process. It can also be seen the interface between the parts with distinct dopants. To evaluate the dyes distribution in the microstructures, we obtained fluorescence confocal microscopy images of the double doped microstructure, displayed in Fig. 2(b) (false colors). Such image shows that the dyes are distributed throughout the microstructure. It is important to observe that, even after repeated washing procedures (inherent to the approach described here), the microstructures maintain their structural features and there is no mixing of dopants. The colors in Fig. 2(b) were applied with the aid of software, based on an analysis of the fluorescence microscopy image.

 

Fig. 2 (a) Scanning electron micrograph of a double-doped microstructure (top view). (b) Confocal fluorescent microscopy image of the same microstructure.

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The black dotted-line in Fig. 3 shows the emission spectrum of the double-doped microstructure presented in Fig. 2, in which two emission peaks at approximately 535 and 580 nm are observed. Such peaks correspond, respectively, to the emission of Disodium Fluorescein and Rhodamine B. The red line in Fig. 3 represents the two-peak Gaussian fit, where it is clear the contribution of the two dopants contained in the microstructure to its total emission.

 

Fig. 3 Emission spectrum of the double-doped microstructure (black) and corresponding two-peak Gaussian fit.

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Since microstructures doped with two dyes have different emission peaks, we can select the emission of the microstructures using different excitation wavelengths and filters, as illustrated in Fig. 4 . Figure 4(a) shows a bright-field image (top view) of a set of double doped microstructures. When light at 350 nm is used to excite the microstructures (Fig. 4(b)), we observe a fluorescence coming out from both parts of the microstructure (with distinct dyes). However, when excitation at 550 nm is employed (Fig. 4(c)), there is no fluorescence of the microstructure part doped with Disodium Fluorescein, because this dye does not absorb at 550 nm, but we see fluorescence only from the part containing Rhodamine B.

 

Fig. 4 (a) Transmission microscope image of double-doped microstructure. (b) Microscope image when excitations at (b) 325 nm and (c) 550 nm are employed.

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This technique does not limit the shape or the number of doped parts of the microstructures which may be produced. Figure 5(a) shows a SEM image of double-doped microstructures containing Disodium Fluorescein and Rhodamine B at its outer and inner part, respectively. The confocal fluorescence image of one of these microstructures is displayed in Fig. 5(b). Again, we observe the interface between the parts of the structures with good structural integrity. We can also observe that the dopants are distributed throughout the microstructure and were not mixed to each other during fabrication nor after washing procedures.

 

Fig. 5 (a) Scanning electron micrograph of double-doped microstructures. (b) Confocal microscope image of the same microstructure.

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We also fabricated a microstructure with three distinct dopants. In this case, besides using Rhodamine B and Disodium Fluorescein, we also used Stilbene 420. Figure 6(a) presents the emission spectrum of a triple doped microstructure (black line). Three peaks can be observed in the emission spectrum, which are related to the emission of each one of the dyes contained in the microstructure. As it can be seen in Fig. 6(a), the experimental data can be fitted by three Gaussian curves centered at 420 nm (Stilbene 420 – dark blue curve), 535 mm (Disodium Fluorescein – green curve) and 580 nm (Rhodamine B – red curve).

 

Fig. 6 (a) Fluorescence spectrum of triple-doped microstructure (black) and corresponding three-peak Gaussian fit (orange). The blue, green and red lines represent, respectively, the Gaussian contributions of Stilbene 420, Disodium Fluorescein and Rhodamine B. (b) Fluorescence of the microstructure when excitation is directed to the region doped with Stilbene 420 (blue) and with Rhodamine B (red).

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In Fig. 6(b) we observe the emission of the triple-doped microstructure obtained by exciting different spatial regions of the sample. The blue curve shows the collected fluorescence spectrum when the excitation is performed at the portion doped with Stilbene 420, while the red curve corresponds to the spectrum obtained when excitation is focused at the Rhodamine B doped part of the microstructure. This result shows that one can select the microstructure region of interest, by exciting/collecting only the desired emission, which is interesting for applications where multiple doping is required. For instance, through the method presented here, RGB fluorescent microdevices could be fabricated. However, this technique is not restricted to applications in optics. This approach is promising for the fabrication of microenvironments to study bacterial or cellular growth, opening new venues for advanced drug delivery systems.

4. Conclusion

We report multi-doped microstructures fabricated via 2PP, containing Rhodamine B, Disodium Fluorescein and Stilbene 420. The microstructured samples display good structural integrity and a broad fluorescent emission. We demonstrate that such emission can be altered by either changing the excitation wavelength or by choosing the sample’s excitation position. This approach can be exploited to design multi-doped microstructured devices aiming at applications in optics and photonics.