1.1 Introduction
In 1990s, the advent of ultrafast laser technology has enabled the observation of the nonlinear optical processes that occur when electric field interacts strongly with materials. With superior features such as deeper penetration depth, lower photobleaching, and minimum invasion, multiphoton excitation (MPE) fluorescence microscopy is particularly suitable for imaging thick tissue and living animals [1]. Additionally, second harmonic generation (SHG), another phenomenon of nonlinear optics, can be employed to directly obtain contour information of non-centrosymmetry within specimens without labeling [2]. However, MPE is not only for the applications of nonlinear optical imaging but also very useful in fabrication. In recent years, MPE microfabrication has become one of the most popular three-dimensional (3D) microfabrication techniques. The utilization of a short pulse width from a femtosecond laser and tight focusing by a high numerical aperture (NA) objective lens are critical for inducing sufficient two-photon absorption (TPA) and for achieving high precision fabrication. Because TPA is confined to the focal volume, microstructures with the desired 3D submicron features can be created [3-5].
Recently, photopolymerization or photocrosslinking based on MPE has also been developed to further improve fabrication resolution since it uses low molecular weight photoinitiators/photoactivators to trigger reactions and requires an optical energy threshold to initiate the photochemistry process [6-8].
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Because multiphoton absorption is more confined to the focal volume, this approach not only allows the creation of finer structures beyond the capability of conventional single-photon lithography, but also provides greater spatial resolution than other 3D microfabrication techniques. As such, multiphoton microfabrication has attracted widespread interest for its potential use in fabricating intrinsic 3D microstructures with sub-diffraction limited spatial resolution [3]. Previously, femtosecond 3D microfabrication has been demonstrated in resin- [3,8], protein- [7], and metal-substrates [9].
Photopolymerization by TPA encompasses a broad range of applications such as microfluidic systems [10], 3D optical storage devices [11], and photonic crystal structures [12]. In addition, when the fs laser peak power is large enough, this nonlinear process, together with cascade ionization, generates very high concentrations of free electrons in the focal volume, resulting in plasma-mediated ablation of material [13]. In general case, the mechanism can be roughly classified as photothermal reaction and multiphoton-induced ablation. Plasma-mediated ablation is a reaction that the energy level is high enough to tear molecules apart, rather than just drive the electronic transitions that lead to fluorescent relaxation; however, photothermal reaction also damages the adjacent region simultaneously. In order to reduce thermal accumulation, it can be accomplished by decreasing the repetition rate [14] or enhancing the efficiency of multiphoton-induce ionization via adopting a shorter ultrafast pulse to improves the machining quality [15]. In particular, the applications of fs laser ablation in micromachining [16-18], generation of
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nanoparticles [19], and formation of nanostructures [20] have advanced rapidly in recent years. Although conventional MPE configuration is versatile for many micro/nano-processing applications; the major drawback of the approaches is the point-scanning process, which slows fabrication speed and is limited to laboratory investigation and prototype fabrication. Although using self-assembly of colloidal spheres is workable for the mass-production of photonics crystals [21], it is limited to periodic structures. Other methods such as glancing angle deposition and the combination of nanolithography with alternating-layer deposition can further enhance the complexity of structures [22,23]. However, the fabrication of large-scale freeform microstructures still cannot be achieved. Still another interference method, namely holographic lithography, can make non-periodic structures [24], to obtain the desired intensity pattern, calculation of the phase information is needed in advance.
Recent studies have shown that using simultaneous spatial and temporal focusing techniques can provide widefield and axially-resolved multiphoton imaging [25-29]. The advantage of widefield multiphoton microscopy is that less time is required to capture one frame, enabling rapid frame rates for capturing dynamic events. With a high-speed, high-sensitivity camera and an ultrahigh peak power laser, an imaging rate of a few hundred frames per second can be achieved [30]. Furthermore, this microscopy setup can be modified as a high-throughput multiphoton microfabrication system for the micromachining of microfluidic channels and optically transparent materials with high aspect ratio features [31,32]. The spatiotemporal focusing technique
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uses a diffraction grating to separate frequencies spatially, and then recombines them on the focal plane of an objective lens. Only in that plane do the different frequency components overlap in phase and produce a short, high-peak power pulse, allowing effective MPE to occur. Further, depending on laser beam spot size and system magnification, the widefield and axially-resolved MPE can excite an entire area, which is a definite advantage compared with conventional point scanning MPE. Therefore, this technique provides a solution for high-speed MPE microfabrication and enables high-throughput manufacturing.
In this study, in order to instantly generate 3D freeform polymer microstructures, a multiphoton microfabrication system based on spatiotemporal focusing and patterned excitation has been developed. This system incorporates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) and a digital micromirror device (DMD) generating two-dimensional (2D) designed patterns on the focal plane. 3D freeform trimethylolpropane triacrylate (TMPTA) polymer microstructures using Rose Bengal (RB) as the photoinitiator were created by sequentially superimposing 2D structures while translating the sample stage axially. This approach can provide a greater than three-order increase in fabrication speed as compared to conventional point scanning MPE. In contrast with holographic femtosecond laser processing [33], the system can simultaneously provide additional nonlinear optical images of the fabricated microstructures for real-time 3D inspection.
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However, resin materials fabricated via two-photon polymerization (TPP) are not very suitable for biomedical applications, and so biocompatible microstructures for further biological research are required. It is now well-known that spatial concentration gradients (i.e. gray-level) of bioactive molecules in the extracellular matrix (ECM) play important roles in several areas of cell biology, including morphogenesis, wound healing, and metastasis [34-36]. Several novel optical schemes improving this limitation by using photochemical approaches to covalently link protein molecules to surfaces have been reported. Hence, how to fabricate large-scale freeform gray-level protein structures should be a big issue for the biomedical researches involved in ECM. Compared to TPP, a two-photon crosslinking (TPC) bio-microstructure is more difficult to achieve by only controlling the average fabrication power; herein, another approach, controlling the pulse number of the laser is adopted. To this end, the gated mode operation of the ultrafast amplifier which can control overall pulse number per layer and the gray-level adjustment of the DMD for further adjusting the pulse number locally were both utilized to select the pulse number. Via the above mechanisms, 3D gray-level covalently-linked bovine serum albumin (BSA) microstructures were fabricated by TPC using RB as the photoactivator. Moreover, RB two-photon excited fluorescence (TPEF) can be used as contrast agent [37,38].
Therefore, online 3D inspection of the fabricated BSA microstructures without washing out the unreacted solution can be offered.
Recently, graphene-based materials have become more interesting due to
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their unique high conductivity, chemical stability, optical property, and intrinsic flexibility [39-42]. Graphene oxide (GO) is an oxidization of graphene which has properties of inexpensive, scalable, and good water-soluble compared to those of graphene; hence it has another advantages for further applications.
However, the electrical property of GO is inferior to that of graphene due to its constituent of oxygen functional groups. Nevertheless, GO sheet can be reduced via chemical and physical methods, which can transform GO into reduced graphene oxide (rGO) resulting in the increase of its conductivity.
Some groups have utilized UV-visible laser sources to reduce GO for fabricating microelectronic devices on the GO films by laser direct writing [43, 44]. Also, this reduction process can be achieved by adopting near-infrared fs laser to fabricate GO microstructure [45,46]. To date, these patterning technologies of graphene and GO sheets can be classified into conventional lithography, soft-lithography, and direct writing and its applications including supercapacitor, microelectrode, biosensor and so on [47]. However, to exploit their practical applications, a prerequisite is mass-production of these microelectronic devices with precise and complex micropatterns or architectures [48]. To address this issue, the temporal focusing configuration described above with pattern illumination can be utilized for high-throughput multiphoton-induced reduction and ablation of GO sheets to achieve GO-based arbitrary micropatterns. The functions of DMD are not only for generating 2D designed patterns, but also to locally control the pulse number via its gray level selection. As the result, the reduction degree of GO patterns could be
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manipulated by pulse number control. In addition, ablation process can also be realized by using the same system, making it potential for mass production of GO-based microcircuit in the further. Furthermore, the size of the pattern is scaled by the magnification of the system; hence, increasing feature size does not require sacrificing patterning time.
1.2 Motivation
Over the past few decades, the ability to guide and direct the growth of axons with engineered precision is an on-going endeavor with broad implications for many diverse areas of research. Experimental research using traditional biological techniques has provided valuable information regarding the neuronal response to individual guidance cues. However, the local environment that growing nerves face is inherently complex and contains a rich mixture of cues whose collective influence on growing nerves is not completely understood. Biomedical engineers and neuroscientists have employed tissue engineering techniques to model the complex in vivo environment of the nervous system as a means of isolating and studying the specific interactions between these cues and the neurons on which they act.
By using MPE fabrication, scalable patterns comprised of different widths and pitch, as well as gradients can be fabricated on the same substrate, allowing for hypothesis testing of the effects of these morphologic factors.
Additionally, the response of axonal outgrowth and polarity to multiple ECM proteins and other adhesive components can also be interrogated.
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Although MPE laser scanning method has optical sectioning capability by using nonlinear optical responses of materials, the sequential nature of raster scanning is inherently slower than parallel process. As the results, the concept of temporal focusing was proposed to overcome this issue. Previously, temporal focusing technique with a fixed optical mask realized MPE microfabrication [49]. The mask was placed at the image-conjugate plane of the grating surface and the focal plane of the objective lens, and so any kind of 3D structure could be created by sequentially changing the optical mask layer-by-layer. However, the use of static optical masks limits fabrication speed;
and since the light source is typically a Ti:sapphire (ti-sa) ultrafast oscillator, the fabrication area is further limited by the available peak power. In this thesis, in order to enhance the fabrication speed and area, ultrafast amplifier system integrated with a DMD has been developed, which makes MPE more suitable for high-throughput fabrication of polymer microstructures, gray-level bio-structures, and GO-based micropatterns.
1.3 Outline
In this thesis, chapter 1 introduces the development of the MPE technique and its applications for biomedical imaging, microfabrication and plasma-mediated ablation. Chapter 2 describes fabrication of 2D micropatterns and 3D microstructures for the guidance of neural growth which were created by conventional laser scanning MPE configuration.
Chapter 3 introduces the concept of temporal focusing excitation at first, and
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then using this setup to fabricate 3D freeform polymer microstructures. The enhancement of fabrication throughput and the function of online inspection are discussed as well. Chapter 4 shows the multiple gray-level BSA biostructures can be fabricated simultaneously via laser pulse control. The concentration of the fabricated BSA structures can be online examined using TPEF of RB as contrast agent. Chapter 5 describes high throughput MPE reduction and ablation of 2D GO thin film. The reduction and machining quality under different experimental condition has been studied. Chapter 6 is the conclusions.
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