Mask-Directed Multiphoton Lithography

Abstract

We report a strategy for rapidly generating arbitrary microscale patterns of biomaterials that can be used to create complex surface gradients or sequentially assembled into functional three-dimensional microarchitectures. In this approach, mask objects and transparency-based masks are used to direct multiphoton photocrosslinking (MPP) of proteins. Unlike conventional methods for controlling direct-write multiphoton fabrication, 1 this simple graphical-based approach allows new patterns to be created within minutes, enabling rapid iteration and optimization of experimental parameters. We demonstrate use of this strategy to transfer detailed shapes (e.g., the silhouette of a house fly) into biomaterial patterns and to fabricate microchambers capable of trapping and incubating a single motile bacterium.Multiphoton lithography provides high-resolution microfabrication capabilities in three dimensions, using nonlinear excitation to promote photopolymerization 2 or protein photocrosslinking 3,4 within a femtoliter voxel created by tightly focusing a pulsed laser beam. By scanning the fabrication voxel through reagent solution, complex three-dimensional microscale objects 1,5 have been produced with lateral feature sizes as small as ∼120 nm using synthetic materials 1 and ∼250 nm using proteins. 4 Figure 1A demonstrates our approach for mask-directed multiphoton lithography. Here, light from a femtosecond titanium: sapphire (76 kHz) laser is sent through an unmodified confocal scan box to raster the beam in a rectangular pattern at a focal plane positioned between the scan box and an inverted microscope. This focal plane (mask plane) is conjugate with the front focal plane of the microscope objective. By placing an object (such as Musca domestica, Figure 1A) or a pattern printed on transparency film within the mask plane, microstructures representing the object silhouette or mask negative can be fabricated at the objective focal plane. Fabrication objects, created by multiphoton crosslinking of bovine serum albumin (BSA) or other proteins, display feature sizes of F mask /M obj , where F mask is the feature size in the mask and M obj is the magnification of the objective/lens system. Minimum achievable feature sizes are determined by the spatial confinement of multiphoton absorption and the diffusion distances of reactive intermediates. 6 From a practical standpoint, masks printed on standard low-resolution transparencies (1200 dpi) can be used with a conventional 100X (1.3 NA) objective to produce features of ∼0.5 µm ( Figure 1A-C)sa scale appropriate for addressing a broad range of applications in cell biology and microtransport. 7 Using this general approach, multiple masks can be used in concert to fabricate complex patterns containing gradients of crosslinked protein ( Figure 1B). Here, a negative transparency photomask was placed in the mask plane to define the microstructure shape and a second fully opaque mask was translated during scanning at a constant velocity to vary the integrated laser exposure across th...

Multiphoton fabrication of chemically responsive protein hydrogels for microactuation

“…We and others have shown that direct-write protein matrices can retain ligand-binding and catalytic functionality similar to that of their component proteins (23)(24)(25)(26)(27), implying that native electrostatic and hydrophobic interactions governing protein conformation remain significantly intact within microstructures. However, at pH extremes, unfavorable electrostatic interactions can disrupt the native state and result in protein unfolding (31,32).…”

“…Moreover, matrices can be cycled rapidly between swelled and condensed states in response to pH steps (Movie S3), indicating (as predicted) that denaturation is reversible. Importantly, the degree to which hydrogel microstructures swell and actuate was found to depend strongly on the crosslinking density of the matrix, a property that can be modulated both by protein concentration within the fabrication solution and laser exposure times (25,26,34) (Fig. S1).…”

“…We previously have shown that protein-based microchambers are well suited for capture and growth of motile cells such as Escherichia coli (26), in part because the porosity of crosslinked protein matrices provides efficient transfer of nutrients and waste materials across microchamber walls. It is possible to trap individual bacteria (26) and to grow up a genetically homogeneous population to extremely high densities (Ϸ10 11 cells per ml) within these chambers.…”

“…In addition to having the potential to undergo shape and hydration changes in response to chemical triggers, proteins contain a large number of weak acids and bases, similar to polymers used in pH-responsive hydrogels (5). Importantly, matrices composed of functional photocrosslinked proteins can be localized in 3D microenvironments by using multiphoton fabrication (23)(24)(25)(26)(27). Thus, multiphoton-fabricated protein structures could provide high-resolution 3D control over the topography of a stimuliresponsive material-facilitating greater mechanical functionality and incorporation of responsive hydrogels into more complex 3D devices-while potentially providing specificity (e.g., through ligand binding) over volumetric responses.…”

Polymers to direct cell fate by controlling the microenvironment

“…Increased 3D resolution down to the submicron range can be achieved with multiphoton photocrosslinking, but at the expense of fabrication time. To decrease processing time, mask-based methods for rapid prototyping have been developed [20] and could be further improved if used in tandem with a micro-mirror based masking device [21]. The utility of microfabrication techniques has been demonstrated in the design of 3D skin substitutes with an artificial epidermis containing micrometer scale features similar to the rete ridges and dermal papillae found in normal skin at the epidermal-dermal junction [22].…”