Multiphoton excited polymerization has attracted increasing attention as a powerful 3 dimensional nano/microfabrication tool. The nonlinear excitation confines the fabrication region to the focal volume allowing the potential to achieve freeform fabrication with submicron capabilities. We report the adaptation and use of a computer aided design (CAD) approach, based on rapid prototyping software, which exploits this potential for fabricating with protein and polymers in biologically compatible aqueous environments. 3D structures are drawn in the STL format creating a solid model that can be sliced, where the individual sections are then serially fabricated without overwriting previous layers. The method is shown for potential biological applications including microfluidics, cell entrapment, and tissue engineering
©2006 Optical Society of America
In the last several years, Multiphoton Excited (MPE) fabrication has emerged as a powerful 3D nano/microfabrication technique. The method is analogous to the more common multiphoton excited fluorescence imaging of live cells and tissues where optical sectioning is obtained by confinement of the excitation to the plane of focus. In terms of nano/microfabrication, MPE photochemistry can be used create 3-D objects one plane at a time, without overwriting previous layers. [1–9] Most of this work has centered on microfabrication with polymer resins, with the research being directed at 3-D storage and nanophotonics applications. [10–12]. However, there are also a wide range of potential applications in biology, where these include tissue engineering, cell sorting by microfluidics, and biosensing. MPE can play a unique role here since more traditional methods such as photolithography and microcontact printing do not have freeform capabilities in all 3 dimensions and have somewhat limited biocompatibility. Additionally, solid freeform fabrication cannot provide the submicron feature sizes necessary for current challenges in tissue engineering, nor does it possess the degree of biocompatibility that is achievable by MPE photochemistry in an aqueous environment. To take advantage of these possibilities, several biological applications using MPE polymerization have recently been demonstrated in the areas of cell biology  and biomaterials [13–14].
We have primarily directed the MPE fabrication technology towards tissue engineering applications and demonstrated that soluble and structural proteins could be crosslinked, layer by layer, into 3-D protein matrices. Through these efforts we have shown that we could precisely and reproducibly control materials properties such as the crosslink density, and corresponding diffusion coefficients.  In addition, excellent biocompatibility was achieved in all cases to date [16–19] For example, we find high levels of specificity and spreading of cells adhered to MPE patterned surfaces, where the method allows for the systematic investigation of the 3-D topographic and biochemical aspects that are important in cell-matrix interactions. Since the MPE crosslinking process affords 3-D nano/microfabrication capabilities, this is an ideal method to create scaffolds directly from the native extracellular matrix (ECM) proteins since biomaterial surfaces can be modified by the addition of crosslinked bioactive species in virtually any desired topography with the appropriate biochemical signals. In this work, we take steps towards advancing the fabrication technology to achieve this goal.
The main advantage in the MPE technique relative to other fabrication methods lies within its potential freeform capabilities and concurrent nano/microscale capabilities. A major challenge in the field has been implementing scanning methods to fully exploit this power. Many reports have used x-y-z stage scanning, as coupled with the appropriate control, allows user-defined fabrication in all 3 dimensions. However, this method is 2–3 orders of magnitude slower than laser-scanning through galvanometer mirrors. The latter is highly preferred for biological applications, where both the starting materials and resulting structure can readily suffer photodamage from prolonged exposure to the high peak powers required for efficient excitation. Additionally, we have previously shown that fabrication rate for proteins is 2–3 orders of magnitude slower than for polymers. However since MPE fabrication is typically a threshold process, simply decreasing the pulse energy with longer pixel dwell times is not a tenable approach to this fabrication task. Thus repetitive laser galvo scanning with freeform capabilities would be the ideal approach.
While laser scanning with galvo mirrors provides great speed, these devices are not designed for complete random access to create user defined patterns. For example, the galvos and control software in laser scanning confocal microscopes are optimized for simple scanning patterns of lines and rectangles. To overcome this limitation, there have been several reports of approaches used to increase the flexibility of laser scanning for MPE fabrication. For example, we constructed an instrument that is capable of variety of geometrical patterns including polygons, ellipses, and circles, in both perimeter and solid patterns. In an alternate approach, Braun  and coworkers use modulated raster scanning, where by controlling the power during the scan with an electro-optic modulator, much greater flexibility was obtained. Maruo  utilized a combination of stage and circular laser scanning to construct an impressive range of micromachines including gears and actuators.
It is of great value for a range of biological applications to extend these ideas and utilize rapid galvo scanning to fabricate user-defined 3D objects directly from soluble proteins. In this article we describe our continuing efforts to achieve this goal, where we link rapid prototyping (RP) stereolithography software into our LabVIEW control code (described in Ref. . RP utilizes the STL file format, which creates a triangular mesh of the surface of a solid to be fabricated. This method has been widely used in a variety of industries (e.g. automotive, aerospace, and medical device industries) to create macroscale (mm-cm) physical objects directly from CAD data sources. In this process any desired structure or combination of structures of user-defined complexity and intricacy can be drawn and then rapidly fabricated. Here we adapt this 3D fabrication method to biological applications by crosslinking proteins from a freely diffusing aqueous environment. The overall approach begins with 3D solid CAD drawing that is sliced into individual files, where these are fed to our laser scanning fabrication microscope. We show several examples of the method, including devices for cell sorting, cell encapsulation, and tissue engineering.
2. Experimental methods
2.1. Materials and methods
Octadecyltrichlorosilane (Gelest), anhydrous toluene (Aldrich), type I and type II collagen (Sigma), Bovine Serum Albumin (BSA), Rose Bengal (Sigma) were used as received. Water soluble ethoxylated trimethylolpropane triacrylate (TMPTA) was kindly provided by Sartomer.
We have described the MPE photochemistry for crosslinking proteins and polymers in detail previously. Briefly, we use two-photon excitation of Rose Bengal (1 mM) at 780 nm. Polymerization of the TMPTA requires a co-initiator, where 0.1M of TEA is used. Crosslinking proteins requires only the Rose Bengal photoactivator. BSA was used at a concentration of 10 mg/mL, or ~10-4M. Preparations were created on silanized microscope slides under cover slips that were separated from the slide using glass spacers, such that the enclosed volume was approximately 50 µL.
2.3. Fabrication instrument/microscope
The laser- scanning fabrication microscope has been described in detail  and is only briefly described here. The multi-photon excitation is achieved through a femtosecond near-infrared titanium-sapphire oscillator (Mira 900-F, Coherent) that is pumped by a 5 W Verdi Nd:YVO4 (Coherent). The laser is coupled to an upright microscope (Zeiss Axioskop,) that is equipped with both bright field and fluorescence optics, which are used as online diagnostics of the fabrication process. Scanning is achieved by a combination of the laser galvo scanning mirrors as well as a programmable x-y-z motorized stage. Typical pulse energies at the sample were approximately 500 pJ/pulse. A 20X, 0.75 numerical aperture (NA) objective lens was used for the fabrication. The code for operating this instrument for both the scan control and simultaneous data acquisition was written entirely using LabVIEW 7 (National Instruments) and is freely available on our website at:
Two-photon fluorescence is used for imaging the fabricated structures, where the contrast arises from either residual Rose Bengal photoactivator or from staining with Rhodamine B or Di-4-ANEPPS. 3D stacks are acquired on an Olympus Fluoview modified for 2-photon imaging. Rendering of the stacks was performed with Imaris Bitplane.
2.4. CAD STL approach
Our overarching scheme to achieve flexible fabrication through laser scanning is to draw a solid 3D model and convert this into a format that is readable by the LabVIEW code that controls the scanning of the galvo mirrors. A flow chart is shown in Fig. 1, which shows the AutoCad (AutoDesk™) or Pro-E™ In the simplest version, AutoCad can export individual DXF files, which can be processed as described below. Alternatively, RP software can convert a 3D drawing to a solid STL rendering. This model is then sliced into individual DXF files using RP software, where we use Materialise Magics. The resulting slices are then hatched in AutoCAD, where the density of the fill pattern defines the crosslink density. After a hatched DXF file is created, an executable program written in LabVIEW transforms the raw coordinate data from the DXF file into a voltage array that is sent to the galvanometers. The crosslink density of the structure can be controlled by defining the fill pattern in AutoCAD, where the choosing a tight fill pattern will directly correlate to denser polymerization or crosslinking. This parameter can then be spatially defined over the region of the scan to provide rapid, true 3D fabrication control. Alternatively, the slices can be singly or cross-hatched, depending on the desired structural considerations.
Here we demonstrate our CAD-based MPE fabrication in a microscopic aqueous environment by presenting several examples of biological applications using the STL format to fabricate objects from proteins and polymers. As a demonstrative example, we fabricated a microflow “tunnel” from a water soluble triacrylate. Solid renderings of four of the 20 STL slices are shown in Fig. 2(b). The resulting structure has dimensions of 130 µm (l) by 100 µm (w) by 90 µm (h), with wall thickness 10 µm. Following MPE polymerization, the chamber was washed with water three times to remove un-reacted monomer as well as any entrapped Rose Bengal photoactivator. The structure was stained with Rhodamine B and imaged by two-photon excited fluorescence (TPEF) at 830 nm. Figure 2 shows two views of the 3D rendered fluorescence image, where (b) and (c) show the two openings as well as the curved interior.
This type of structure is not possible to create by lithographic methods or by conventional geometric laser scanning. It should be possible to use this fabrication technology to create microflow devices for sorting cells. As a rudimentary example, we have placed L1210 lymphocytes inside a MPE fabricated tunnel (325×140×172 µm). The structure was washed as described above. The cells were stained with the membrane staining dye Di-4-ANNEPS and introduced into the tunnel by microinjection, such that the cells flowed in a gentle vortex into the tunnel. A 3D TPEF stack was obtained and a one optical section near the bottom of the tunnel, containing the cells is shown in the left panel of Fig. 3, where the arrows point to the cells (diameter ~10 microns). The right panel shows an optical section near the middle region of the structure and cells are seen near the entrance. This type of structure could further be functionalized with cell attractive molecules such as fibronectin, collagen, or growth factors.
3.1. MPE crosslinked protein structures
While we demonstrated the CAD based control of the fabrication process with a water soluble acrylate, we ultimately wish to exploit the technology for biological applications where fabricated devices can be created from proteins. Here we show several simple examples to demonstrate the functionality of the approach. Figure 4(a) shows the 3D rendering of the TPEF image of a high aspect ratio cylinder fabricated from MPE crosslinked BSA, where the height is 60 µm, opening of 60 µm, and wall thickness of 1µm. This form of structure can be used to entrap cells to perform motility migration and studies. To this end as shown in Fig 4(b), we have a fabricated a cylinder from a mixture of crosslinked Texas Red labeled fibrinogen and BSA, where the former provides ECM cues through RGD binding sites, and the latter is used to provide increased mechanical stability. The inner rectangular matrix was fabricated from the same mixture. The image in Fig. 4(b) shows two GFP expressing neurons (isolated from primary culture) that have migrated into the contained region and have attached to the inner matrix. A fibroblast at the right edge has also migrated towards this region. The general problems of cell motility and migration are not well understood and other work has reported the use of cell entrapment as a means to gain insight into these phenomena. We suggest that MPE fabricating the entrapment vessel as well functionalizing its interior may be a versatile tool to study motility and migration on attractive surfaces in a well-controlled, spatially confined environment.
The CAD based MPE fabrication can also be used to create tissue engineering scaffolds. In previous work, we have studied cell spreading and morphology on simple MPE crosslinked linear patterns, and found that crosslinked ECM proteins effectively direct cell growth.[18–19] We suggested this occurs because these structures provide both topographic and ECM cues that replicate aspects of the native ECM. Here we extend to this to a more complex scaffold. Figure 5(a) shows the 3D rendering of a scaffold created from a 50/50% mixture of Texas Red labeled fibrinogen and BSA, where the mesh size is 10 microns. The image was shadowed to better show the 3D architecture. Figure 5(b) shows a GFP expressing fibroblast adhered to the surface of the scaffold as well as more cells approaching the structure at the 4 hour timepoint. This result shows that the structure possessed its bioactivity in terms of providing ECM cues for cell-binding.
While the scaffold shown in Fig. 5 has more 3D structure than we have previously reported, native ECMs have significantly more complex topography. Our long-term goals are to use the freeform capabilities of MPE fabrication to replicate this structure. As a step in this direction, we can use the CAD control approach to fabricate complex, arbitrary 3D structures.
As an example, Fig. 6 shows the process for fabricating the University of Connecticut oak leaf logo. A CAD drawing was created from a JPEG image, processed in AutoCAD and Magics as described above (Fig. 6 left). The logo was then fabricated from BSA, where an optical section of the 3D fluorescence image stack and 3D rendering are shown in the middle and right panels, respectively. The structure is approximately 100 microns in diameter, and faithfully reproduces the original drawing. In the future, we will use this approach to fabricate topographically complex tissue engineering scaffolds.
4. Discussion and conclusions
MPE nano/microfabrication has great potential in biological applications in areas including tissue engineering and biosensing, as well as a tool for probing fundamental questions in cell biology. Given the slow crosslinking rates as well as low damage thresholds relative to polymers, there is a large premium on the speed of fabrication while utilizing the lowest exposure doses possible. The method described here affords 3D capabilities with good speed. For example, the cell-sorting tunnel (Fig. 2) and the UCONN Oak leaf (Fig. 6) required less than a minute of fabrication time. While we specifically used AutoCAD and Materialise to link to our open source LabVIEW code, we point out that the method would be applicable with any software capable of producing STL output files.
There have been other recent reports of using rapid prototyping software for tissue engineering applications.[26, 27] However, these approaches have used either conventional commercially available STL machines, or direct light projection using standard components. Since these approaches are based on the standard methodologies, the resulting minimum feature sizes are in the range of a few hundred microns to several mm. By contrast the technology reported here adapts the powerful CAD based RP approach to the submicron and micron scale and performs the fabrication with biological materials in a biocompatible aqueous environment. This opens up the door to an array of applications in tissue engineering, biosensing, and cell biology.
We gratefully acknowledge support under an NIH 1U54-RR022232.
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