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Optical Materials Express

Optical Materials Express

  • Editor: David J. Hagan
  • Vol. 2, Iss. 12 — Dec. 1, 2012
  • pp: 1803–1808
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Emission features of microstructures fabricated by two-photon polymerization containing three organic dyes

A. J. G. Otuka, V. Tribuzi, D. S. Corrêa, and C. R. Mendonça  »View Author Affiliations


Optical Materials Express, Vol. 2, Issue 12, pp. 1803-1808 (2012)
http://dx.doi.org/10.1364/OME.2.001803


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Abstract

Fabrication of microstructures containing active compounds, such as fluorescent dyes and nanoparticles have been exploited in the last few years, aiming at applications from photonics to biology. Here we fabricate, using two-photon polymerization, microstructures containing the fluorescent dyes Stilbene 420, Disodium Fluorescein and Rhodamine B. The produced microstructures, containing dyes at specific sites, present good structural integrity and a broad fluorescence spectrum, from about 350 nm until 700 nm. Such spectrum can be tuned by using different excitation wavelengths and selecting the excitation position in the microstructure. These results are interesting for designing multi-doped structures, presenting tunable and broad fluorescence spectrum.

© 2012 OSA

1. Introduction

In the last few years, the 2PP fabrication of microstructures containing compounds of interest has been demonstrated [6

6. C. R. Mendonça, D. S. Correa, F. Marlow, T. Voss, P. Tayalia, and E. Mazur, “Three-dimensional fabrication of optically active microstructures containing an electroluminescent polymer,” Appl. Phys. Lett. 95(11), 113309 (2009). [CrossRef]

8

8. J. Wang, H. Xia, B. B. Xu, L. G. Niu, D. Wu, Q. D. Chen, and H. B. Sun, “Remote manipulation of micronanomachines containing magnetic nanoparticles,” Opt. Lett. 34(5), 581–583 (2009). [CrossRef] [PubMed]

], with interesting prospects for the development of applications [8

8. J. Wang, H. Xia, B. B. Xu, L. G. Niu, D. Wu, Q. D. Chen, and H. B. Sun, “Remote manipulation of micronanomachines containing magnetic nanoparticles,” Opt. Lett. 34(5), 581–583 (2009). [CrossRef] [PubMed]

,9

9. D. A. Parthenopoulos and P. M. Rentzepis, “Three-dimensional optical storage memory,” Science 245(4920), 843–845 (1989). [CrossRef] [PubMed]

]. 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

10. A. Doraiswamy, C. Jin, R. J. Narayan, P. Mageswaran, P. Mente, R. Modi, R. Auyeung, D. B. Chrisey, A. Ovsianikov, and B. Chichkov, “Two photon induced polymerization of organic-inorganic hybrid biomaterials for microstructured medical devices,” Acta Biomater. 2(3), 267–275 (2006). [CrossRef] [PubMed]

,11

11. P. Tayalia, C. R. Mendonça, T. Baldacchini, D. J. Mooney, and E. Mazur, “3D Cell-Migration Studies using Two-Photon Engineered Polymer Scaffolds,” Adv. Mater. 20(23), 4494–4498 (2008). [CrossRef]

].

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

12. A. Žukauskas, M. Malinauskas, L. Kontenis, V. Purlys, D. Paipulas, M. Vengris, and R. Gadonas, “Organic Dye Doped Microstructures for Optically Active Functional Devices Fabricated via Two-Photon Polymerization Technique,” Lith. J. Phys. 50(1), 55–61 (2010). [CrossRef]

]. 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

13. D. S. Correa, M. R. Cardoso, V. Tribuzi, L. Misoguti, and C. R. Mendonça, “Femtosecond Laser in Polymeric Materials: Microfabrication of Doped Structures and Micromachining,” IEEE J. Sel. Top. Quantum Electron. 18(1), 176–186 (2012). [CrossRef]

].

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

14. T. Baldacchini, C. N. LaFratta, R. A. Farrer, M. C. Teich, B. E. A. Saleh, M. J. Naughton, and J. T. Fourkas, “Acrylic-based resin with favorable properties for three-dimensional two-photon polymerization,” J. Appl. Phys. 95(11), 6072–6076 (2004). [CrossRef]

]. The monomers, together with the photoinitiator (Lucirin TPO-L) [14

14. T. Baldacchini, C. N. LaFratta, R. A. Farrer, M. C. Teich, B. E. A. Saleh, M. J. Naughton, and J. T. Fourkas, “Acrylic-based resin with favorable properties for three-dimensional two-photon polymerization,” J. Appl. Phys. 95(11), 6072–6076 (2004). [CrossRef]

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

15. A. K. Sharma, D. S. Ahlawat, D. Mohan, and R. D. Singh, “Concentration-dependent energy transfer studies in ternary dye mixture of Stilbene-420, Coumarin-540 and Nile Blue,” Spectrochim. Acta A Mol. Biomol. Spectrosc. 71(5), 1631–1633 (2009). [CrossRef] [PubMed]

17

17. R. F. Kubin and A. N. Fletcher, “Fluorescence quantum yields of some rhodamine dyes,” J. Lumin. 27(4), 455–462 (1982). [CrossRef]

], 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.

The scanning electron microscope (SEM) image (top view) of a double doped microstructure (Disodium Fluorescein and Rhodamine B) is presented in Fig. 2(a)
Fig. 2 (a) Scanning electron micrograph of a double-doped microstructure (top view). (b) Confocal fluorescent microscopy image of the same microstructure.
. 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.

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

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
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.
. 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.

This technique does not limit the shape or the number of doped parts of the microstructures which may be produced. Figure 5(a)
Fig. 5 (a) Scanning electron micrograph of double-doped microstructures. (b) Confocal microscope image of the same microstructure.
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.

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.

Acknowledgments

The authors acknowledge financial support from FAPESP, CNPq and CAPES from Brazil. Technical assistance from André L. S. Romero is gratefully acknowledged.

References and links

1.

A. Ovsianikov, X. Shizhou, M. Farsari, M. Vamvakaki, C. Fotakis, and B. N. Chichkov, “Shrinkage of microstructures produced by two-photon polymerization of Zr-based hybrid photosensitive materials,” Opt. Express 17(4), 2143–2148 (2009). [CrossRef] [PubMed]

2.

S. Maruo and S. Kawata, “Two-photon-absorbed near-infrared photopolymerization for three-dimensional microfabrication,” J. Microelectromech. Syst. 7(4), 411–415 (1998). [CrossRef]

3.

M. P. Joshi, H. E. Pudavar, J. Swiatkiewicz, P. N. Prasad, and B. A. Reianhardt, “Three-dimensional optical circuitry using two-photon-assisted polymerization,” Appl. Phys. Lett. 74(2), 170–172 (1999). [CrossRef]

4.

Y. H. Pao and P. M. Rentzepis, “Laser-Induced Production of Free Radicals in Organic Compounds (77 Degrees K Polymerization of Styrene and P-Isobutylstyrene 2-Photon Absorption E),” Appl. Phys. Lett. 6(5), 93–95 (1965). [CrossRef]

5.

S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett. 22(2), 132–134 (1997). [CrossRef] [PubMed]

6.

C. R. Mendonça, D. S. Correa, F. Marlow, T. Voss, P. Tayalia, and E. Mazur, “Three-dimensional fabrication of optically active microstructures containing an electroluminescent polymer,” Appl. Phys. Lett. 95(11), 113309 (2009). [CrossRef]

7.

V. Tribuzi, D. S. Corrêa, W. Avansi Jr, C. Ribeiro, E. Longo, and C. R. Mendonça, “Indirect doping of microstructures fabricated by two-photon polymerization with gold nanoparticles,” Opt. Express 20(19), 21107–21113 (2012). [CrossRef] [PubMed]

8.

J. Wang, H. Xia, B. B. Xu, L. G. Niu, D. Wu, Q. D. Chen, and H. B. Sun, “Remote manipulation of micronanomachines containing magnetic nanoparticles,” Opt. Lett. 34(5), 581–583 (2009). [CrossRef] [PubMed]

9.

D. A. Parthenopoulos and P. M. Rentzepis, “Three-dimensional optical storage memory,” Science 245(4920), 843–845 (1989). [CrossRef] [PubMed]

10.

A. Doraiswamy, C. Jin, R. J. Narayan, P. Mageswaran, P. Mente, R. Modi, R. Auyeung, D. B. Chrisey, A. Ovsianikov, and B. Chichkov, “Two photon induced polymerization of organic-inorganic hybrid biomaterials for microstructured medical devices,” Acta Biomater. 2(3), 267–275 (2006). [CrossRef] [PubMed]

11.

P. Tayalia, C. R. Mendonça, T. Baldacchini, D. J. Mooney, and E. Mazur, “3D Cell-Migration Studies using Two-Photon Engineered Polymer Scaffolds,” Adv. Mater. 20(23), 4494–4498 (2008). [CrossRef]

12.

A. Žukauskas, M. Malinauskas, L. Kontenis, V. Purlys, D. Paipulas, M. Vengris, and R. Gadonas, “Organic Dye Doped Microstructures for Optically Active Functional Devices Fabricated via Two-Photon Polymerization Technique,” Lith. J. Phys. 50(1), 55–61 (2010). [CrossRef]

13.

D. S. Correa, M. R. Cardoso, V. Tribuzi, L. Misoguti, and C. R. Mendonça, “Femtosecond Laser in Polymeric Materials: Microfabrication of Doped Structures and Micromachining,” IEEE J. Sel. Top. Quantum Electron. 18(1), 176–186 (2012). [CrossRef]

14.

T. Baldacchini, C. N. LaFratta, R. A. Farrer, M. C. Teich, B. E. A. Saleh, M. J. Naughton, and J. T. Fourkas, “Acrylic-based resin with favorable properties for three-dimensional two-photon polymerization,” J. Appl. Phys. 95(11), 6072–6076 (2004). [CrossRef]

15.

A. K. Sharma, D. S. Ahlawat, D. Mohan, and R. D. Singh, “Concentration-dependent energy transfer studies in ternary dye mixture of Stilbene-420, Coumarin-540 and Nile Blue,” Spectrochim. Acta A Mol. Biomol. Spectrosc. 71(5), 1631–1633 (2009). [CrossRef] [PubMed]

16.

T. D. Z. Atvars, C. A. Bortolato, and D. Dibbern-Brunelli, “Electronic absorption and fluorescence spectra of xanthene dyes in polymers,” J. Photochem. Photobiol. Chem. 68(1), 41–50 (1992). [CrossRef]

17.

R. F. Kubin and A. N. Fletcher, “Fluorescence quantum yields of some rhodamine dyes,” J. Lumin. 27(4), 455–462 (1982). [CrossRef]

OCIS Codes
(160.2540) Materials : Fluorescent and luminescent materials
(230.4000) Optical devices : Microstructure fabrication

ToC Category:
Laser Materials Processing

History
Original Manuscript: October 3, 2012
Revised Manuscript: November 7, 2012
Manuscript Accepted: November 15, 2012
Published: November 26, 2012

Citation
A. J. G. Otuka, V. Tribuzi, D. S. Corrêa, and C. R. Mendonça, "Emission features of microstructures fabricated by two-photon polymerization containing three organic dyes," Opt. Mater. Express 2, 1803-1808 (2012)
http://www.opticsinfobase.org/ome/abstract.cfm?URI=ome-2-12-1803


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References

  1. A. Ovsianikov, X. Shizhou, M. Farsari, M. Vamvakaki, C. Fotakis, and B. N. Chichkov, “Shrinkage of microstructures produced by two-photon polymerization of Zr-based hybrid photosensitive materials,” Opt. Express17(4), 2143–2148 (2009). [CrossRef] [PubMed]
  2. S. Maruo and S. Kawata, “Two-photon-absorbed near-infrared photopolymerization for three-dimensional microfabrication,” J. Microelectromech. Syst.7(4), 411–415 (1998). [CrossRef]
  3. M. P. Joshi, H. E. Pudavar, J. Swiatkiewicz, P. N. Prasad, and B. A. Reianhardt, “Three-dimensional optical circuitry using two-photon-assisted polymerization,” Appl. Phys. Lett.74(2), 170–172 (1999). [CrossRef]
  4. Y. H. Pao and P. M. Rentzepis, “Laser-Induced Production of Free Radicals in Organic Compounds (77 Degrees K Polymerization of Styrene and P-Isobutylstyrene 2-Photon Absorption E),” Appl. Phys. Lett.6(5), 93–95 (1965). [CrossRef]
  5. S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett.22(2), 132–134 (1997). [CrossRef] [PubMed]
  6. C. R. Mendonça, D. S. Correa, F. Marlow, T. Voss, P. Tayalia, and E. Mazur, “Three-dimensional fabrication of optically active microstructures containing an electroluminescent polymer,” Appl. Phys. Lett.95(11), 113309 (2009). [CrossRef]
  7. V. Tribuzi, D. S. Corrêa, W. Avansi, C. Ribeiro, E. Longo, and C. R. Mendonça, “Indirect doping of microstructures fabricated by two-photon polymerization with gold nanoparticles,” Opt. Express20(19), 21107–21113 (2012). [CrossRef] [PubMed]
  8. J. Wang, H. Xia, B. B. Xu, L. G. Niu, D. Wu, Q. D. Chen, and H. B. Sun, “Remote manipulation of micronanomachines containing magnetic nanoparticles,” Opt. Lett.34(5), 581–583 (2009). [CrossRef] [PubMed]
  9. D. A. Parthenopoulos and P. M. Rentzepis, “Three-dimensional optical storage memory,” Science245(4920), 843–845 (1989). [CrossRef] [PubMed]
  10. A. Doraiswamy, C. Jin, R. J. Narayan, P. Mageswaran, P. Mente, R. Modi, R. Auyeung, D. B. Chrisey, A. Ovsianikov, and B. Chichkov, “Two photon induced polymerization of organic-inorganic hybrid biomaterials for microstructured medical devices,” Acta Biomater.2(3), 267–275 (2006). [CrossRef] [PubMed]
  11. P. Tayalia, C. R. Mendonça, T. Baldacchini, D. J. Mooney, and E. Mazur, “3D Cell-Migration Studies using Two-Photon Engineered Polymer Scaffolds,” Adv. Mater.20(23), 4494–4498 (2008). [CrossRef]
  12. A. Žukauskas, M. Malinauskas, L. Kontenis, V. Purlys, D. Paipulas, M. Vengris, and R. Gadonas, “Organic Dye Doped Microstructures for Optically Active Functional Devices Fabricated via Two-Photon Polymerization Technique,” Lith. J. Phys.50(1), 55–61 (2010). [CrossRef]
  13. D. S. Correa, M. R. Cardoso, V. Tribuzi, L. Misoguti, and C. R. Mendonça, “Femtosecond Laser in Polymeric Materials: Microfabrication of Doped Structures and Micromachining,” IEEE J. Sel. Top. Quantum Electron.18(1), 176–186 (2012). [CrossRef]
  14. T. Baldacchini, C. N. LaFratta, R. A. Farrer, M. C. Teich, B. E. A. Saleh, M. J. Naughton, and J. T. Fourkas, “Acrylic-based resin with favorable properties for three-dimensional two-photon polymerization,” J. Appl. Phys.95(11), 6072–6076 (2004). [CrossRef]
  15. A. K. Sharma, D. S. Ahlawat, D. Mohan, and R. D. Singh, “Concentration-dependent energy transfer studies in ternary dye mixture of Stilbene-420, Coumarin-540 and Nile Blue,” Spectrochim. Acta A Mol. Biomol. Spectrosc.71(5), 1631–1633 (2009). [CrossRef] [PubMed]
  16. T. D. Z. Atvars, C. A. Bortolato, and D. Dibbern-Brunelli, “Electronic absorption and fluorescence spectra of xanthene dyes in polymers,” J. Photochem. Photobiol. Chem.68(1), 41–50 (1992). [CrossRef]
  17. R. F. Kubin and A. N. Fletcher, “Fluorescence quantum yields of some rhodamine dyes,” J. Lumin.27(4), 455–462 (1982). [CrossRef]

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