As one of the few 3D microfabrication techniques available to researchers, multiphoton lithography (MPL) has generated considerable interest in the scientific community. By allowing researchers to localize photochemistry to a femtoliter volume, MPL has permitted the fabrication of intricate, 3D microstructures from a range of materials, including protein hydrogels. MPL can be used to fabricate functional hydrogels on the scale of 100 μm, with features on the order of 1 μm. This dissertation examines existing MPL techniques to discover ways in which current processes can be modified to produce hydrogel products that are more useful for biomedical applications like tissue engineering. A new material is introduced that enables the fabrication of fully unconstrained hydrogel microstructures. In this context, A structure can be classified as “unconstrained” when it is free to translate and rotate without hindrance in three dimensions, and is not attached to the substrate or any other structure. New processes are demonstrated that permit the fabrication of larger MPL hydrogels without sacrificing feature resolution. This allows the fabrication of millimeter-scale, high aspect ratio structures with features smaller than 10 μm. Methods are described for tuning and measuring the mechanical properties of MPL-fabricated hydrogels, and ways of tuning the functional properties of the hydrogels are also examined. / text

This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus. / text

Dynamic materials which can sense changes in their surroundings and subsequently respond or adapt by autonomously altering their functionality, surface chemistry, transparency, color, wetting behavior, adhesiveness, shape, etc. are primed to be integral components of future "smart" technologies. However, such systems can be quite complex and often require intricate coordination between both chemical and mechanical inputs/outputs as well as the combination of multiple materials working cooperatively to achieve the proper functionality. It is critical to not only understand the fundamental behaviors of existing dynamic chemo-mechanical systems, but also to apply that knowledge and explore new avenues for design of novel materials platforms which could provide a basis for future adaptive technologies. Part 1 explores the use of environmentally-sensitive hydrogels, either alone or within arrays of high-aspect-ratio nano/microstructures, as chemo-mechanical actuators. Chapters 1 through 7 describe a bio-inspired approach to the design of hybrid actuating surfaces in which the volume-changing hydrogel acts as the “muscle” that reversibly actuates the microstructured "bone". In particular, the different actuation mechanisms arising from variations in how the hydrogel is integrated into the structure array, how chemical signals can be used to manipulate actuation parameters, and finally how such a system may be used for applications ranging from adaptive optics to manipulation of chemical reactions are described. Chapter 8 discusses the use of responsive hydrogel scaffolds as a means to mechanically compress cells and direct differentiation. Part II explores dynamic microsystems involving the integration of catalytic sites within intricately structured 3D microenvironments. Specifically, we explore a generalizable and straightforward route to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. The catalytic, electrical, and electrochemical properties are characterized, and we demonstrate high resolution integration of catalysts within 3D-defined microenvironments to generate directed particle and fluid transport. / Chemistry and Chemical Biology

Master of Science / Department of Chemistry / Daniel A. Higgins / Lithography techniques have been widely used to fabricate optical, electronic and optoelectronic devices with sub-micron scale spatial resolution. In the most common lithographic procedures, a light sensitive polymer, called a photoresist, is exposed and developed to form a binary relief pattern on a substrate. The finest features are produced by X-ray or electron-beam methods, both of which are very expensive to employ. Less expensive methods use ultraviolet (UV) light to expose the photoresist through a photomask. The resolution in these methods is somewhat lower and is governed by diffraction of light by the photomask, the quality of the photomask, and by any chemical/physical development steps subsequently employed. Due to the above limitations, we have been investigating direct-write, ablative multiphoton lithography as an alternative method for preparing high-resolution patterns. With this method, near-IR light from an ultrafast pulsed laser source is focused into a polymer film, leading to depolymerization and vaporization of the polymer. Arbitrary binary patterns can be produced by raster scanning the sample while controlling exposure of the film to the laser. Importantly, high-resolution etching of the polymer film is achieved without the use of a photomask and without chemical development steps. While arbitrary patters are easily prepared, it is also possible to prepare three-dimensional (i.e. grayscale) surface relief structures. In this study, ablative multiphoton photolithography is used to prepare binary and grayscale structures in thin films of PEDOT:PSS, an electrically conductive organic polymer blend. A simple kinetic model is proposed to explain the etching process. Data on the power-dependence of polymer etching can be fit to this model and is used to determine the order of the nonlinear optical process involved. The etch depth as a function of laser focus is also investigated and shown to follow the same kinetic model. The results show that three-dimensional (grayscale) patterns can be prepared by modulating either the laser power or the laser focus. Images of several binary and grayscale structures prepared by this method are presented.