Diffractive maskless lithography uses computer-generated holograms that are displayed on a phase-only SLM to shape and distribute coherent laser light in three dimensions. As shown in , the SLM is positioned at a focal plane conjugate to the pupil plane (back focal plane) of a high numerical aperture (NA) microscope objective. The phase distributions displayed on the SLM, commonly termed phase holograms, are derived from the Gerchberg-Saxton algorithm (GSA) [20
] and generate 2D light distributions by adjusting the phase of a uniform wavefront. The Fourier transforming property of a lens converts the angular information contained within the modified wavefront into the desired spatial distribution(s) at the focal plane. illustrates the conversion of a phase hologram derived from a target template into an intensity distribution at the focal plane of the system. Similar to projection methods this distribution can be used to perform lithography. However, while the intensity distribution reflects an accurate representation of the target image, discontinuities comprised of zero intensity result from the presence of speckle noise. A time-averaged corrective technique [16
] previously used in photographic films was modified and implemented to improve pattern continuity for the single-shot patterning mode.
In order to perform single-shot lithography, several phase holograms containing the phase information for a desired image were calculated and displayed in succession. The stochastic nature of the phase error introduced during the calculation of each phase hologram, which causes speckle noise, was averaged by the exposure of a region to multiple iterations of the same computer-generated hologram. The fidelity of the final pattern can be expressed in terms of the speckle contrast,
is the standard deviation,
is the average intensity, and N
is the number of hologram additions. Equation (1)
reveals that the image contrast can be reduced by increasing the number of hologram additions. The single-shot capabilities of the system were evaluated by varying the N
value within a preset exposure time of 10 s. Several N
values were selected to understand the trade-off between increased image fidelity and hologram calculation time. A significant improvement in overall image fidelity was realized using as few as 10 holograms, C
= 0.32. Further evaluation suggested that an exposure containing 30 holograms (C
= 0.18), requiring less than 35 seconds to calculate, provided highly resolved and continuous patterns.
The versatility of the maskless single-shot mode was demonstrated by the fabrication of patterns within S-1813 positive photoresist. A diverse range of arbitrary templates were designed and patterned on a single sample within 10 minutes (
) using single-shot holographic processing. The templates can be designed with any image processing software capable of creating bitmaps, jpegs, or tiffs. Resolutions of approximately 700 nm are achieved in patterns that contain features with at least one single-pixel dimension . The Rayleigh criterion predicts a minimum resolution of 232 nm for the system (532 nm wavelength, 1.4 NA); however, the non-ideal properties of the SLM, including a 19 µm pixel pitch and 93% fill factor, likely account for this discrepancy in resolution. Nevertheless, these results indicate that the phase-based single-shot mode is capable of providing resolution and pattern continuity comparable to many amplitude-based OPL methods.
Fig. 2 SEM images of various 2D patterns fabricated using the described method with a 100x objective. The inset SEM images captured at a 26° angle to the sample surface show the photoresist depth. The patterns in (a-e) were fabricated using the single-shot (more ...)
The parallel processing capabilities of the system were also demonstrated using S-1813 resist. A user-defined feature was created and discretized into pixel coordinates. Next, individual holograms containing phase information for an array of foci were generated for each of these coordinates. User-defined spacing and feature size, along with the FOV of the microscope objective, dictate the number of features/foci in the final multi-component pattern. When displayed on the SLM, each phase hologram produces an intensity distribution containing the specified number of arrayed foci. Because the SLM has a frame rate of 72 Hz, the holograms may be combined and displayed sequentially as a movie for serial patterning. The serial fabrication of a 25-feature array of squares  using this methodology took less than a minute.
Having demonstrated the versatility of the 2D serial and single-shot modes, we next attempted fabrication of 3D microstructures. Because the linear absorption characteristics of the S-1813 at the 532 nm laser wavelength preclude 3D fabrication, a commercially available photopolymer (Norland 63) was used. The absorption properties of the polymer and the nonlinear excitation provided by the laser enable the 3D positioning of voxels. A specially calculated lens phase [21
] was added to each hologram to shift 2D distributions into arbitrary planes. The axial shifts required for a layer-by-layer 3D fabrication approach were determined by the depth of focus (DOF) within the target material. The DOF in the photopolymer, which was experimentally determined to be ~5 µm, dictated the axial resolution during fabrication. However, the axial resolution could be improved when fabricating solid structures without voids by adjusting the axial shifts such that the layers overlap.
through show nine different microstructures that were simultaneously fabricated using multiple foci. These results demonstrate 3D control over the morphology of the fabricated structures as well as the ability to simultaneously fabricate structures with different shapes on the same substrate using a single phase-modulated source beam. We believe that the ability to simultaneously fabricate 3D microstructures of different shapes is a unique feature of this methodology, as we are not aware of another method that provides this capability.
Fig. 3 SEM micrographs of 3D microstructures fabricated using the serial processing mode. (a) The 9-feature pattern was designed in CAD software and then all features were simultaneously fabricated in NOA 63 using a single phase-modulated source beam (
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To further illustrate our 3D fabrication capability, we used the negative tone photoresist SU-8 2010 because the thermal and chemical properties of this epoxy-based resist are better suited for higher aspect ratio micropatterning than the Norland 63. through display SEM micrographs of a microscale bridge that was serially fabricated using two focal points directed by phase holograms derived from a predesigned template . We chose this microstructure to demonstrate the ability of diffractive maskless lithography to fabricate microstructures that contain voids. Because the system utilizes an inverted microscope, the voids beneath the rails demonstrate true 3D fabrication, as the laser light can be focused through areas of the resist leaving them unaltered. This level of spatial control is akin to the many multi-photon systems presented in the literature, but does not require the use of any moving components, such as micropositioning stages.
The noncontact fabrication afforded by diffractive maskless lithography is well suited for the processing of substrates immersed in an aqueous environment, which makes it an attractive method for creating proteinaceous microstructures. Recently, microstructures composed of proteins have been utilized to direct cell motility [22
], detect biological molecules [23
], and as scaffolds for tissue engineering applications [24
]. Towards the goal of creating biosensors based on the ambient analyte theory [25
], we next explored the single-shot fabrication of protein features on the order of tens of microns. A biosensor of this size satisfies the ambient analyte theory because it performs detection of a target in solution such that the equilibrium concentration of the test sample is unaffected. Patterns were created on a glass surface from bovine serum albumin (BSA) conjugated with fluorescein isothiocyanate (FITC) contained within a 20 µL sample well. A three-shape template was transferred to the BSA protein using a time averaged single-shot exposure [
]. The subsequent fluorescent image  and SEM micrograph  indicate the successful fabrication of active microstructures. We believe that the formation of microstructures that are stable in an aqueous environment is due to the photocrosslinking of the protein within the exposure volume mediated by the conjugated FITC, as previously reported by Cremer, Shear, and associates [26
]. Next, to conclusively demonstrate that protein activity is retained after the photocrosslinking of protein microstructures, the retention of biotin binding was tested using a FITC-BSA solution spiked with FITC-avidin. The BSA/avidin structures shown in were incubated with a fluorescently labeled biotin solution and then thoroughly rinsed to remove any unbound biotin. The fluorescent images in and indicate the presence of bound biotin only within the boundaries of the BSA/avidin features. This result clearly demonstrates that protein structures microfabricated by dynamic maskless lithography retain the ability to bind their ligand in an aqueous environment, suggesting that this microfabrication methodology will allow the fabrication of functionally active microstructures of other ligand-binding proteins and enzymes with micron-level spatial resolution.
Fig. 4 BSA-FITC solution exposed to three time-averaged patterns simultaneously at 110 mW for 10 s. (a) Real-time microscope image captured during fabrication. (b) Fluorescent image of patterned protein after sample wash with 1x PBS. (c) SEM image of sample (more ...)
At this point, it is important to point out that the lithographic technique is not limited to operating with only the 532 nm wavelength. While the versatility to fabricate structures in the particular photoresists, photopolymers, and proteins available to our group governed the choice of this wavelength, the SLM can accommodate wavelengths ranging from 400 to 700 nm. By simply replacing the source laser, the system can be tailored to fabricate structures in a variety of photoactive materials.