多光子激發技術因其吸收範圍可侷限於一極小體積內，因此相比於傳統的單光子激發，擁有較佳的切片特性，可製造出高空間解析的任意形狀結構。而近年來，石墨烯（graphene）因其突破性的光、電、機械與生物等特性，成為備受矚目的材料之一。本論文以超快雷射系統結合非線性多光子激發技術，利用氧化石墨烯量子點（graphene oxide quantum dot，GOQD）及其他材料之光化學反應進行三維光微影製程，藉由調控飛秒雷射加工功率、掃描速率以及點與點之間距等參數，製作各式擁有不同應用潛能之微結構。
實驗中，首先利用雙光子聚合反應製作高分子微結構，使用之材料為市售之光學樹脂Norland Optical Adhesive 81。完成之結構展現了其複雜、窄線寬且可自由站立的特性，擁有在光子積體電路中連接晶片的潛力。接著，在論文中呈現了單純蛋白質，以及具GOQD與蛋白質混合的三維微結構。此機制係利用光敏劑之雙光子吸收與激發，產生高活性的單態氧，進而誘發蛋白質分子或GOQD間進行交聯反應。過程中以孟加拉玫瑰素（rose Bengal，RB）作為光敏劑，也稱為光活化劑；牛血清蛋白（bovine serum albumin，BSA）作為交聯之蛋白質。其中GOQD在水溶液中表面帶負電荷，會與RB帶一正電之鈉離子（Na+）相吸而產生聚集效應甚至結晶。此處，BSA除了扮演結構支撐的角色，亦作為分散劑，利用其分子對GOQD的靜電排斥特性，使GOQD受RB影響的聚集效應降至最低，得以長時間穩定且均勻地分散於水溶液中。而在雷射加工方面，為了防止光造成的熱效應破壞結構，並使效率最大化，雷射波長選擇以使RB產生更高的雙光子吸收為主。最後，以調控之最佳參數完成二維與三維的微結構製作，並透過掃描式電子顯微鏡、白光影像和雙光子激發螢光影像檢視結構的表面形態。

Since multiphoton excitation (MPE) can be confined to the focal volume, this technique provides better sectioning effect compared to conventional single-photon excitation, which can be used to create arbitrary three-dimensional (3D) structures of high spatial resolution. Besides, graphene has become a popular material in recent years due to its unique electronic, mechanical, thermal, optical, and biological properties. In this thesis, the technique of nonlinear MPE based on femtosecond laser scanning system was utilized to conduct 3D photolithography through photochemical reactions in graphene oxide quantum dot (GOQD) and other materials. By adjusting the fabrication parameters such as laser power, scanning speed, scanning times per layer, and the overlap of the points, various microstructures owning potentials for different applications were created.
In this study, microstructures of polymer produced using two-photon polymerization is first presented. The employed material is a commercially available liquid resin, Norland Optical Adhesive 81. The fabricated structures show the complexity, narrow linewidth, and ability of free-standing, which is promising in photonic integrated circuit. Next, 3D microstructures of protein and GOQD-protein composite are demonstrated. The mechanism is crosslinking induced by multiphoton absorption and MPE of the photosensitizer, which generates reactive singlet oxygen that make protein molecules or GOQDs bond together. In the process, rose Bengal (RB) acts as the photosensitizer, or called photoactivator; bovine serum albumin (BSA) acts as the crosslinked protein. Particularly, GOQDs are negatively surface-charged in aqueous solution, resulting in adsorption to Na ions (Na+) of RB. This will make a problem of serious aggregation or even crystallization. Herein, BSA serves as not only the support of the structures but also the dispersant. The repulsion of its molecule to GOQD can effectively alleviate the aggregation caused by RB, making the solution stably disperse for a long period. In the fabrication process, laser wavelength is chosen depending on the two-photon absorption spectrum of RB to prevent the thermal effect and for the optimal efficiency. At last, we accomplished two-dimensional and 3D microstructures with optimized parameters, and observed the morphology by scanning electron microscope, bright-field and two-photon excited fluorescence imaging.

[1] J. D. Bhawalkar, G. S. He, and P. N. Prasad, “Nonlinear multiphoton processes in organic and polymeric materials,” Rep. Prog. Phys. 59, 1041-1070 (1996).
[2] M. Göppert-Mayer, “Elementary processes with two quantum transitions,” Ann. Phys. 18, 466-479 (2009).
[3] W. Kaiser and C. G. B. Garrett, “Two-photon excitation in CaF2:Eu2+,” Phys. Rev. Lett. 7, 229-231 (1961).
[4] S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett. 22, 132-134 (1997).
[5] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, and A. A. Firsov, “Electric field effect in atomically thin carbon films,” Science 306, 666-669 (2004).
[6] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, M. I. Katsnelson, I. V. Grigorieva, S. V. Dubonos, and A. A. Firsov, “Two-dimensional gas of massless Dirac fermions in graphene,” Nature 438, 197-200 (2005).
[7] A. K. Geim and K. S. Novoselov, “The rise of graphene,” Nat. Mater. 6, 183-191 (2007).
[8] C. Lee, X. Wei, J. W. Kysar, and J. Hone, “Measurement of the elastic properties and intrinsic strength of monolayer graphene,” Science 321, 385-388 (2008).
[9] A. A. Balandin, S. Ghosh, W. Bao, I. Calizo, D. Teweldebrhan, F. Miao, and C. N. Lau, “Superior thermal conductivity of single-layer graphene,” Nano Lett. 8, 902-907 (2008).
[10] D. Li and R. B. Kaner, “Graphene-based materials,” Science 320, 1170-1171 (2008).
[11] D. A. Dikin, S. Stankovich, E. J. Zimney, R. D. Piner, G. H. B. Dommett, G. Evmenenko, S. T. Nguyen, and R. S. Ruoff, “Preparation and characterization of graphene oxide paper,” Nature 448, 457-460 (2007).
[12] G. Eda, G. Fanchini, and M. Chhowalla, “Large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material,” Nat. Nanotechnol. 3, 270-274 (2008).
[13] X. Sun, Z. Liu, K. Welsher, J. T. Robinson, A. Goodwin, S. Zaric, and H. Dai, “Nano-graphene oxide for cellular imaging and drug delivery,” Nano Res. 1, 203-212 (2008).
[14] Y. Zhou, Q. Bao, B. Varghese, L. A. L. Tang, C. K. Tan, C.-H. Sow, and K. P. Loh, “Microstructuring of graphene oxide nanosheets using direct laser writing,” Adv. Mater. 22, 67-71 (2010).
[15] H. Chen, M. B. Müller, K. J. Gilmore, G. G. Wallace, and D. Li, “Mechanically strong, electrically conductive, and biocompatible graphene paper,” Adv. Mater. 20, 3557-3561 (2008).
[16] N. A. Kotov, J. O. Winter, I. P. Clements, E. Jan, B. P. Timko, S. Campidelli, S. Pathak, A. Mazzatenta, C. M. Lieber, M. Prato, R. V. Bellamkonda, G. A. Silva, N. W. S. Kam, F. Patolsky, and L. Ballerini, “Nanomaterials for neural interfaces,” Adv. Mater. 21, 3970-4004 (2009).
[17] D. R. Dreyer, S. Park, C. W. Bielawski, and R. S. Ruoff, “The chemistry of graphene oxide,” Chem. Soc. Rev. 39, 228-240 (2010).
[18] J.-Y. Hong and J. Jang, “Micropatterning of graphene sheets: recent advances in techniques and applications,” J. Mater. Chem. 22, 8179-8191 (2012).
[19] J. D. Pitts, P. J. Campagnola, G. A. Epling, and S. L. Goodman, “Submicron multiphoton free-form fabrication of proteins and polymers: studies of reaction efficiencies and applications in sustained release,” Macromolecules 33, 1514-1523 (2000).
[20] S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature 412, 697-698 (2001).
[21] M. Miwa, S. Juodkazis, T. Kawakami, S. Matsuo, and H. Misawa, “Femtosecond two-photon stereo-lithography,” Appl. Phys. A: Mater. Sci. Process 73, 561-566 (2001).
[22] T. Watanabe, M. Akiyama, K. Totani, S. M. Kuebler, F. Stellacci, W. Wenseleers, K. Braun, S. R. Marder, and J. W. Perry, “Photoresponsive hydrogel microstructure fabricated by two-photon initiated polymerization,” Adv. Funct. Mater. 12, 611-614 (2002).
[23] Z. B. Sun, X. Z. Dong, W. Q. Chen, S. Nakanishi, M. Duan, and S. Kawata, “Multicolor polymer nanocomposites: in situ synthesis and fabrication of 3D microstructures,” Adv. Mater. 20, 914-919 (2008).
[24] A. Marcinkevicius, S. Juodkazis, M. Watanabe, M. Miwa, S. Matsuo, and H. Misawa, “Femtosecond laser-assisted three-dimensional microfabrication in silica,” Opt. Lett. 26, 277-279 (2001).
[25] P. W. Wu, W. C. Cheng, I. B. Martini, B. Dunn, B. J. Schwartz, and E. Yablonovitch, “Two-photon photographic production of three-dimensional metallic structures within a dielectric matrix,” Adv. Mater. 12, 1438-1441 (2000).
[26] Y. Y. Cao, N. Takeyasu, T. Tanaka, X. M. Duan, and S. Kawata, “3D metallic nanostructure fabrication by surfactant-assisted multiphoton-induced reduction,” Small 5, 1144-1148 (2009).
[27] F. Bonaccorso, L. Colombo, G. Yu, M. Stoller, V. Tozzini, A. C. Ferrari, R. S. Ruoff, and V. Pellegrini, “Graphene, related two-dimensional crystals, and hybrid systems for energy conversion and storage,” Science 347, 1246501 (2015).
[28] D. R. Cooper, B. D’Anjou, N. Ghattamaneni, B. Harack, M. Hilke, A. Horth, N. Majlis, M. Massicotte, L. Vandsburger, E. Whiteway, and V. Yu, “Experimental review of graphene,” ISRN Condens. Matter Phys. 2012, 1-56 (2012).
[29] J. I. Paredes, S. Villar-Rodil, A. Martinez-Alonso, and J. M. D. Tascón, “Graphene oxide dispersions in organic solvents,” Langmuir 24, 10560-10564 (2008).
[30] B. M. Gillette, J. A. Jensen, B. Tang, G. J. Yang, A. Bazargan-Lari, M. Zhong, and S. K. Sia, “In situ collagen assembly for integrating microfabricated three-dimensional cell-seeded matrices,” Nat. Mater. 7, 636-640 (2008).
[31] S. Chen, J. Zhu, X. Wu, Q. Han, and X. Wang, “Graphene oxide-MnO2 Nanocomposites for supercapacitors,” ACS Nano 4, 2822-2830 (2010).
[32] K. P. Loh, Q. Bao, G. Eda, and M. Chhowalla, “Graphene oxide as a chemically tunable platform for optical applications,” Nat. Chem. 2, 1015-1024 (2010).
[33] H. A. Becerril, J. Mao, Z. Liu, R. M. Stoltenberg, Z. Bao, and Y. Chen, “Evaluation of solution-processed reduced graphene oxide film as transparent conductors,” ACS Nano 2, 463-470 (2008).
[34] Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts, and R. S. Ruoff, “Graphene and graphene oxide: synthesis, properties, and applications,” Adv. Mater. 22, 3906-3924 (2010).
[35] Y. Zhou, Q. Bao, B. Varghese, L. A. L. Tang, C. K. Tan, C.-H. Sow, and K. P. Loh, “Microstructuring of graphene oxide nanosheets using direct laser writing,” Adv. Mater. 22, 67-71 (2010).
[36] Y. Zhang, L. Guo, S. Wei, Y. He, H. Xia, Q. Chen, H.-B. Sun, and F.-S. Xiao, “Direct imprinting of microcircuits on graphene oxides film by femtosecond laser reduction,” Nano Today 5, 15-20 (2010).
[37] L. Guo, H.-B. Jiang, R.-Q. Shao, Y.-L. Zhang, S.-Y. Xie, J.-N. Wang, X.-B. Li, F. Jiang, Q.-D. Chen, T. Zhang, and H.-B. Sun, “Two-beam-laser interference mediated reduction, patterning and nanostructuring of graphene oxide for the production of a flexible humidity sensing device,” Carbon 50, 1667-1673 (2012).
[38] G. R. Souza, J. R. Molina, R. M. Raphael, M. G. Ozawa, D. J. Stark, C. S. Levin, L. F. Bronk, J. S. Ananta, J. Mandelin, M.-M. Georgescu, J. A. Bankson, J. G. Gelovani, T. C. Killian, W. Arap, and R. Pasqualini, “Three-dimensional tissue culture based on magnetic cell levitation,” Nat. Nanotechnol. 5, 291-296 (2010).
[39] S. R. Shin, H. Bae, J. M. Cha, J. Y. Mun, Y.-C. Chen, H. Tekin, H. Shin, S. Farshchi, M. R. Dokmeci, S. Tang, and A. Khademhosseini, “Carbon nanotube reinforced hybrid microgels as scaffold materials for cell encapsulation,” ACS Nano 6, 362-372 (2012).
[40] M. Kalbacova, A. Broz, J. Kong, and M. Kalbac, “Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells,” Carbon 48, 4323-4329 (2010).
[41] S. Y. Park, J. Park, S. H. Sim, M. G. Sung, K. S. Kim, B. H. Hong, and S. Hong, “Enhanced differentiation of human neural stem cells into neurons on graphene,” Adv. Mater. 23, H263-H267 (2011).
[42] T. R. Nayak, H. Andersen, V. S. Makam, C. Khaw, S. Bae, X. Xu, P.-L. R. Ee, J.-H. Ahn, B. H. Hong, G. Pastorin, and B. Özyilmaz, “Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells,” ACS Nano 5, 4670-4678 (2011).
[43] W. C. Lee, C. H. Y. X. Lim, H. Shi, L. A. L. Tang, Y. Wang, C. T. Lim, and K. P. Loh, “Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide,” ACS Nano 5, 7334-7341 (2011).
[44] J. Park, S. Park, S. Ryu, S. H. Bhang, J. Kim, J.-K. Yoon, Y. H. Park, S.-P. Cho, S. Lee, B. H. Hong, and B.-S. Kim, “Graphene-regulated cardiomyogenic differentiation process of mesenchymal stem cells by enhancing the expression of extracellular matrix proteins and cell signaling molecules,” Adv. Healthc. Mater. 3, 176-181 (2014).
[45] K. Zhang, H. Zheng, S. Liang, and C. Gao, “Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth,” Acta Biomater. 37, 131-142 (2016).
[46] J.-T. Jeong, M.-K. Choi, Y. Sim, J.-T. Lim, G.-S. Kim, M.-J. Seong, J.-H. Hyung, K. S. Kim, A. Umar, and S.-K. Lee, “Effect of graphene oxide ratio on the cell adhesion and growth behavior on a graphene oxide-coated silicon substrate,” Sci. Rep. 6, 33835 (2016).
[47] M. Lorenzoni, F. Brandi, S. Dante, A. Giugni, and B. Torre, “Simple and effective graphene laser processing for neuron patterning application,” Sci. Rep. 3, 1954 (2013).
[48] C. Li and G. Shi, “Three-dimensional graphene architectures,” Nanoscale 4, 5549-5563 (2012).
[49] S. Ushiba, S. Shoji, K. Masui, P. Kuray, J. Kono, and S. Kawata, “3D microfabrication of single-wall carbon nanotube/polymer composites by two-photon polymerization lithography,” Carbon 59, 283-288 (2013).
[50] B. E. A. Saleh and M. C. Teich, Fundamentals of Photonics, 2nd ed., Wiley, Hoboken, NJ (2007).
[51] P. A. Franken, A. E. Hill, C. W. Peters, and G. Weinreich, “Generation of optical harmonics,” Phys. Rev. Lett. 7, 118-119 (1961).
[52] R. W. Boyd, Nonlinear Optics, 3rd ed., Academic Press, Burlington, MA (2008).
[53] B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy, Oxford University Press, New York, NY (2008).
[54] W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73-76 (1990).
[55] A. Gleizes, J. J. Gonzalez, and P. Freton, “Thermal plasma modelling,” J. Phys. D: Appl. Phys. 38, R153-R183 (2005).
[56] F. He, Y. Liao, J. Lin, J. Song, L. Qiao, Y. Cheng, and K. Sugioka, “Femtosecond laser fabrication of monolithically integrated microfluidic sensors in glass,” Sensors 14, 19402-19440 (2014).
[57] W.-S. Kuo, C.-H. Lien, K.-C. Cho, C.-Y. Chang, C.-Y. Lin, L. L. H. Huang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “Multiphoton fabrication of freeform polymer microstructures with gold nanorods,” Opt. Express 18, 27550-27559 (2010).
[58] C. I. Richards, J.-C. Hsiang, and R. M. Dickson, “Synchronously amplified fluorescence image recovery (SAFIRe),” J. Phys. Chem. B 114, 660-665 (2010).
[59] K. Kinoshita, T. Saito, A. Ito, T. Kawakami, Y. Kitagawa, S. Yamanaka, K. Yamaguchi, M. Okumura, “Theoretical study on singlet oxygen adsorption onto surface of graphene-like aromatic hydrocarbon molecules,” Polyhedron 30, 3249-3255 (2011).
[60] W. S. Hummers and R. E. Offeman, “Preparation of graphitic oxide,” J. Am. Chem. Soc. 80, 1339-1339 (1958).
[61] T.-F. Yeh, C.-Y. Teng, S.-J. Chen, and H. Teng, “Nitrogen-doped graphene oxide quantum dots as photocatalysts for overall water-splitting under visible light illumination,” Adv. Mater. 26, 3297-3303 (2014).
[62] D. Li, M. B. Müller, S. Gilje, R. B. Kaner, and G. G. Wallace, “Processable aqueous dispersions of graphene nanosheets,” Nat. Nanotechnol. 3, 101-105 (2008).
[63] S. Ahadian, M. Estili, V. J. Surya, J. Ramón-Azcón, X. Liang, H. Shiku, M. Ramalingam, T. Matsue, Y. Sakka, H. Bae, K. Nakajima, Y. Kawazoe, and A. Khademhosseini, “Facile and green production of aqueous graphene dispersions for biomedical applications,” Nanoscale 7, 6436-6443 (2015).
[64] H.-Y. Chang, C.-Y. Lin, C.-Y. Chang, H. Teng, P. J. Campagnola, and S.-J. Chen, “Graphene oxide dot-based microstructures via dispersion and support of bovine serum albumin,” Opt. Mater. Express 6, 3193-3201 (2016).
[65] Y. Xu, K. Sheng, C. Li, and G. Shi, “Self-assembled graphene hydrogel via a one-step hydrothermal process,” ACS Nano 4, 4324-4330 (2010).