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Abstract
We report on a laser system combining one-photon and two-photon polymerization for precise and fast fabrication of macroscopic three-dimensional structures featuring microscale and nanoscale characteristics. This single-stage process significantly reduces the production time as demonstrated by scaffolds in a shell application. Porous scaffolds with different pore sizes are surrounded by a ring so that cells can be seeded directly to the scaffolds kept in a shell and do not spread over the whole substrate expecting a saving of cell suspension, faster growth on the scaffolds, and a more controllable environment. Compared to a two-photon polymerization process, the ring is fabricated about 500 times faster using one-photon polymerization. The presented hybrid process qualifies for further applications illustrated by a fluidic system.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Additive manufacturing is of great interest for optical, medical, and lab-on-a-chip applications due to its potential for high-resolution, time-saving, and cost-efficient fabrication of three-dimensional structures. Optical techniques enable a variation in light source and exposure parameters leading to an enormous flexibility in the choice of materials and geometry of the structures [1]. Furthermore, there is no need to adhere to layer by layer fabrication using direct laser writing via two-photon absorption to create arbitrary shapes with a precision down to 100 nm [2, 3]. This nonlinear process modifies a photosensitive material only around the high energy density achieved in the focus of an incoming laser beam and, thus, allows pinpoint writing of real three-dimensional structures. The applicability of this method, however, is restricted to small structures subject to a process time of several hours per cubic millimeter [4–6]. By multi-beam parallel processing [7,8] and minimization of the traveling distance during the fabrication [9] the process is further optimized. Additionally, the polymerized volume per beam can be affected by varying the focus condition using objectives with different numerical aperture (NA) adapted to bigger parts and smaller features of the sample [10]. Nevertheless, the point-by-point fabrication nature of this process remains unchanged. For this reason, the combination of two-photon polymerization with other processes comes into focus.
Combining additive and subtractive fabrication was realized by some groups using a single femtosecond pulsed laser source for two-photon polymerization and material ablation [11,12] or material illumination followed by etching [13]. Another approach is based on rapid multiplication of structures fabricated with a two-photon process by micromolding techniques [14,15]. Moreover, fused filament fabrication, a rapid 3D printing technique heating a thermoplastic above its melting point and applying it through a fine nozzle, was employed as a tool to build up samples which are, in a next step, modified by two-photon polymerization [16,17]. By comparison, a one-photon polymerization approach known from stereolithography has the advantage of being an additive optical technique using similar or even the same materials as for two-photon polymerization. The idea of involving one-photon polymerization in the two-photon fabrication process is known from literature. It was used to generate large-area 2D periodic structures with a two-beam interference technique that was subsequently modified by two-photon polymerization [18] or to post-exposure structures where only the surface layers are polymerized by two-photon polymerization [19]. To emphasize the difference to our approach, it is worth to stress that these methods apply one-photon polymerization in a separated process step that is not directly connected to the two-photon exposure step and that these methods are still limited to the high-resolution pinpoint writing characteristics of the two-photon polymerization process. In notable contrast, we introduce a new approach of combining one-photon and two-photon polymerization in a hybrid process that aims at the integration of two flexible 3D-capable methods, using the advantages of both stereolithography and two-photon polymerization, both being flexible 3D technique.
In conventional stereolithography systems, a photosensitive material is solidified at the surface by an ultraviolet light source and a three-dimensional structure is built up layer by layer [20]. Pinpoint writing is also possible and requires additional restrictions regarding the absorption behavior of the material and focusing of the light beam [21,22]. An approach to first fabricate a coarse structure with ultraviolet irradiation and subsequently finer features with two-photon absorption is presented in [23]. This method is limited by the working distance of the focused beam for two-photon polymerization. Given this restriction, an improved layer by layer approach for accurate and fast fabrication applying both optical processes is described in [24] overcoming the working distance limitation.
In this work, we demonstrate, to the best of our knowledge, the first successes in realizing this promising concept [24] of a hybrid polymerization process. A 405 nm continuous wave light source for one-photon polymerization and a 515 nm ultrashort pulse light source for two-photon polymerization are combined in a single system to fabricate macroscopic structures with fine three-dimensional functional elements. We highlight the generation of scaffolds in a shell made from OrmoComp, an organic-inorganic hybrid polymer, being suitable for both absorption processes [9, 25]. Furthermore, the functionality of a channel with a filter system between two glass coverslips is tested as a basis for more complex fluidic applications. The presented structures demonstrate the potential of the hybrid technique that can be of high interest for the fabrication of functional tissue-specific implants with a high-resolution scaffold surface fabricated by two-photon polymerization and a faster fabricated core fabricated by stereolithography [26] or for improving the microchannels of a stereolithography fabricated microfluidic systems like the HEMOS device [27] by additionally applying two-photon polymerization during the fabrication process.
2. Experimental
A hybrid system as shown in Fig. 1(a) is used to create macroscopic structures with microscopic features. An integrated femtosecond fiber laser (Menlo Systems, BlueCut) with a second-harmonic generation extension provides 515 nm laser pulses with a pulse length of 540 fs and a repetition rate of 10 MHz. The laser beam is expanded before it is focused by a 20x objective with an NA of 0.5 (Zeiss, EC Epiplan-Neofluar) mounted on a nanopositioning z-stage (Aerotech, ANT95-50-L-Z). A second beam from a 405 nm cw diode laser (Omicron, Luxx) is also guided through the objective to a sample, which is positioned on a plate movable in the plane perpendicular to the incoming beam using a translation stage (Aerotech, ANT130-XY). The fabrication process is monitored by a CCD camera.
Fig. 1 (a) Fabrication setup. A 515 nm laser beam is polarized with a wave plate (WP), guided through a beam expander, and, such as a 405 nm laser, coupled through an objective movable along the z-axis to a sample fixed on a movable XY stage and (b) process chart illustrating the generation of scaffolds surrounded by a ring.
2.1. Fabrication process
Three-dimensional structures are fabricated into a layer of photosensitive OrmoComp (micro resist technology) sandwiched between to glass coverslips. The coverslips are either spin-coated with OrmoPrime (micro resist technology) to increase the adhesion of a polymerized structure or prepared with an anti-sticking layer described in the following subsection. Figure 1(b) shows exemplary the process to generate scaffolds surrounded by a ring. First, the scaffolds are written on an OrmoPrime coated glass coverslip by two-photon polymerization applying the 515 nm laser and then a ring is added irradiating the sample with the 405 nm laser. This ultraviolet light source cures the entire layer thickness of around 700 µm between the subjacent and upper coverslip so that the ring height is defined by the layer thickness of the polymer. An anti-sticking layer on the upper substrate prevents adhesion and ensures the removal of this coverslip. After production, the structures are cleaned with 2-propanol and distilled water.
2.2. Anti-sticking layer
Anti-sticking sample preparation was performed according to Roos et al. [28]. Before being coated, glass substrates were cleaned with acetone (≧99, 5%), dried in air and then were rinsed in 2-propanol for 5 minutes. In the next step, substrates were immersed in isooctane (CarlRoth, ROTIPURAN≧99,5%, p.a., ACS) for 5 minutes in order to replace 2-propanol at the glass surface. To achieve the anti-sticking self-assembling monolayer, the glass substrates were dipped into a 4 mmol solution of (1,1,2,2 H perfluorooctyl)-trichlorosilane (Sigma Aldrich) in isooctane for 10 minutes at a temperature of −5°C. Residual silane was removed by rinsing the glass substrates for 5 minutes with isooctane, followed by a cleaning procedure in 2-propanol and then in deionized water, 5 minutes in an ultrasonic bath each. Afterwards, the coated glass substrates were blow dried with a commercial blow-dryer. Finally, the substrates were baked under nitrogen atmosphere at 200°C for one hour.
3. Results and discussion
Scaffolds in a shell and channels with integrated filter are successfully produced by combining one-photon and two-photon polymerization in a joint setup. Before both processes are consecutively performed in a prepared sample, the applicability of the chosen wavelengths for the polymerization processes is analyzed. Figure 2(a) shows the result of the measured transmittance spectrum of a sample prepare with OrmoComp identically to the samples for the fabrication process. Bearing in mind that the measured reflection is negligibly small, it clearly reveals that the absorption is significantly higher at 405 nm than at 515 nm. Furthermore, the absorption order for a wavelength of 515 nm was determined using the method presented in [29]. The minimum pulse energy, the threshold energy for fabricating a defined structure in OrmoComp, was identified for several repetition rates with a writing speed of 1 mm/s and 5 mm/s. As demonstrated in Fig. 2(b), all values of the same writing speed are in a straight line by a double-log plot. A fit according to the dependency between the repetition rate frepand threshold pulse energy Eth
yields in an absorption order n of two. Due to these results, it is assumed that there is a one-photon absorption process at 405 nm and a two-photon absorption process at 515 nm.Fig. 2 (a) Measured transmittance of a sample prepare with OrmoComp and (b) dependency between repetition rate and threshold pulse energy.
3.1. Scaffolds in a shell
A single-line single-pass technique [9] is used to fabricate porous and reproducible scaffolds. The thickness of the connections that form a scaffold is kept small for high porosity and thick enough to avoid deformation or even collapse of the structures. This is obtained by modifying the laser power of the 515 nm femtosecond laser chosen for irradiation. The most suitable average laser powers behind the objective to realize different pore sizes from 30 µm up to 110 µm are presented in Fig. 3. A higher stability of the scaffold connections is needed for larger pores correlated with a reduced number of connections for these larger pore sizes. Thus, a higher laser power is necessary to increase the thickness of the connections directly proportional to the pore size. A dependency between pore size and laser power can be determined by considering the calculation of the size of a single voxel [30]. The thickness of the connections is given by the lateral and axial length of a polymerized voxel in the connection. These lengths can be calculated assuming a threshold intensity to initiate the polymerization process and a Gaussian beam intensity distribution
with the threshold intensity Ithand the Gaussian beam intensity distribution I(r, z ) and solving the equations Ith= I(r, 0) and Ith= I(0, z ) for r and z. The intensity at the center I0is proportional to the average laser power P and this leads to the following dependencies between the lengths of a polymerized voxel and the average laser power whereby the determined laser powers in Fig. 3 show a behavior similar to a fit of these to functions.Additionally, an average laser power of 11 µW suffices to fabricate a ring with the 405 nm laser surrounding the scaffolds. This ring is added to improve and better control cell growth on the scaffolds during cell studies. Its aim is to hold cells near the scaffolds for saving cell suspension and faster growth in a restricted environment. The motivation for adding this ring is given by former cell studies with human primary fibroblasts showing that the cells spread over the whole substrate after seeding on the scaffolds. Figure 4 shows that the substrate region around the structure was densely populated after four days (Fig. 4(a)) and that it took nine days (Fig. 4(b)) until the cells have densely populated the upper surface of the scaffold. To accelerate such cell proliferation, spatial confinement around the scaffolds is provided by a ring preventing the cells spreading over the whole substrate.
Fig. 4 Scanning electron microscopy images showing the cell growth on a scaffold and the substrate around the scaffold after four (a) and nine (b) days of cell culture.
Figure 5(a) displays a complete structure of scaffolds and ring taken by a swingable digital microscope (Leica, DVM6). The different scaffold pore sizes are visible by higher magnification (Fig. 5(b)). For further studies, the samples are sputtered with a 15 nm thick gold layer to perform scanning electron microscopy with a Phenom Pro X desktop microscope (Phenom-World). An overview image of scaffolds with the examined pore sizes from 30 µm to 110 µm is provided in Fig. 5(c) and highlights the high resolution of the process by fabricating smaller pore sizes. Figure 5(d) shows the open sidewalls of the scaffolds by a detailed view of a structure with 80 µm pore size. This is achieved by beam expansion and enables a proper perfusion of the scaffolds during cell culture experiments. It is an improvement compared to the results in [9] demonstrating successful horizontal and vertical, inner and outer cell growth on scaffolds with closed sidewalls.
Fig. 5 Scaffolds in a shell structure at different magnifications (a), (b) and scanning electron microscopy images showing scaffolds with the examined pore sizes (c) and the open sidewalls by a detailed view of a structure with 80 µm pore size (d).
The production time of the scaffolds implemented by a nonstop single-line single-pass process using a writing speed of 5 mm/s is significantly shorter compared to other recently published studies on direct laser written scaffolds [6, 31]. In addition, a larger pore size decreases the production time because of the shorter traveling distance during the fabrication and increases the produced volume per minute by a factor of four as presented by the measured values for structures with different pore sizes in Fig. 6(a). This can be explained by looking at two pore sizes, pore size a and pore size b = 2a. The scaffolds are build up by circles in three dimensions as exemplified in Fig. 6(b) by the traveling path during the fabrication and illustrated by a written three-dimensional scaffold with open pores in Fig. 5(d). Instead of one circle per pore surface with diameter b = 2a, there are four circles with diameter a using the smaller pore size. This leads to a factor of two according to the traveling distance. Additionally, to reach a whole scaffold with half of the pore size (a instead of b = 2a), a second scaffold has to be placed between the larger pores with pore size b to reach pores with a pore size of a in all three dimensions. This leads again to a factor of two and in total to a factor of four. The measured value for the smallest pore size of 30 µm deviates from the expected value due to the increasing gap between the desired and actual position and speed of the movable axes for smaller distances of acceleration induced by smaller pore sizes.
Fig. 6 (a) Pore size dependent produced volume per minute and (b) traveling path during the fabrication of scaffolds.
An important point is the time saving brought by using one-photon instead of two-photon polymerization for producing the macroscopic ring with a inner diameter of 6 mm and a ring width of 1 mm. The two-photon process has a point-by-point fabrication nature. This means that the OrmoComp layer with a thickness of around 700 µm is too thick to be polymerized within a single pass using this process and has to be divided into several layers (about fifty layers with the chosen objective). In comparison, the 405 nm laser light is guided through the objective with a beam diameter of 1 mm at the back aperture of the objective reducing the NA of the focused beam. This leads to a wider intensity distribution and a higher aspect ratio. The entire layer thickness is cured without a z-scan using this light source. Furthermore, the ring width of 1 mm has to be sliced into at least ten times more circles to ensure an overlap of adjacent cured circles considering the higher lateral resolution achieved by two-photon polymerization. This results in an enormous production time difference between both processes comparing about 60 hours for two-photon with only 7 minutes for one-photon absorption for a macroscopic ring holding cells near the scaffolds without the need for high resolution.
3.2. Fluidic system
The potential of combining one-photon and two-photon additive manufacturing is further demonstrated by a channel with integrated filter as proof of concept for fluidic systems. A channel is generated into a layer of photosensitive material by ultraviolet light such that it adheres to the subjacent and upper substrate. The geometry of the channel or any chosen geometry is freely adaptable by simply modifying the CAD design. This is an advantage compared to other mask-based photolithography methods that are applicable to polymerize a specific channel or ring [32]. Additonally, a filter is written into the channel applying two-photon polymerization. The production time for a small filter with an overall dimension of 1.6 × 0.4 × 0.96 mm3 and 80 µm pore size is 3.4 min and for a 8 mm long solid channel 3.9 min. The tightness of a fabricated channel is examined by a rhodamine B solution in distilled water and illustrated in Fig. 7(a). Rhodamine B solution flows through the channel and penetrates the filter without any visible leakage in the channel. An enhanced contrast between the transparent fluidic system and the surrounding is obtained by carefully removing the upper coverslip and coloring the system with rhodamine B solution. In Fig. 7(b), a colored channel with filter structure is shown. The creation of tight channels and the option to integrate functional features, as demonstrated by the filter, during a single processing step qualify a hybrid one-photon and two-photon process for specific fluidic applications.
Fig. 7 (a) Rhodamine B solution filled channel and (b) colored system after removing the upper coverslip.
4. Conclusion
In this study, a laser system combining one-photon and two-photon polymerization is introduced. We realized high resolution scaffolds with different pore size and open sidewalls using direct laser writing via two-photon absorption. These scaffolds were surrounded by a fast fabricated ring applying an ultraviolet laser. An anti-sticking layer successfully prevents adhesion of the ring on the upper coverslip. The production time for the ring is reduced significantly from 60 hours for two-photon to 7 minutes for one-photon fabrication. Furthermore, a channel with filter system is realized between two glass coverslips. A tightness examination of the channel with rhodamine B solution and the integration of a functional feature demonstrate applicability of the hybrid process for the generation of fluidic systems. In summary, these results are a promising basis for a fabrication method with several layers forming within each of these layers coarse structures by one-photon polymerization and fine structures by two-photon polymerization.
Funding
German Federal Ministry of Education and Research (BMBF) (Eurostars E!9765 (Hybrid-3D)).
Acknowledgments
This work was supported by the European program “Eurostars” under the project number E!9765 (Hybrid-3D) and funded by the German partner with resources from the German Federal Ministry of Education and Research (BMBF).
Disclosures
The authors declare that there are no conflicts of interest related to this article.
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