Laser-imprinting of micro-3D printed protein hydrogels enables real-time independent modification of substrate topography and elastic modulus.

Independent control over the Young's modulus and topography of a hydrogel cell culture substrate is necessary to characterize how attributes of its adherent surface affect cellular responses. Arbitrary, real-time manipulation of these parameters at the micron scale would further provide cellular biologists and bioengineers with the tools to study and control numerous highly dynamic behaviors including cellular adhesion, motility, metastasis, and differentiation. Although physical, chemical, thermal, and light-based strategies have been developed to influence Young's modulus and topography of hydrogel substrates, independent control of these physical attributes has remained elusive, spatial resolution is often limited, and features commonly must be pre-patterned. We recently reported a strategy in which biomaterials having specified three-dimensional (3D) morphologies are micro-3D printed in a two-step process: laser-scanning bioprinting of a protein-based hydrogel, followed by biocompatible hydrogel re-scanning to create microscale imprinted features at user-defined times. In this approach, a pulsed near-infrared laser beam is focused within the printed hydrogel to promote matrix contraction through multiphoton crosslinking, where scanning the laser focus projects a user-defined topographical pattern on the surface without subjecting the hydrogel-solution interface to damaging light intensities. Here, we extend this strategy, demonstrating the ability to decouple dynamic topographical changes from changes in hydrogel Young's modulus at the substrate surface by increasing the isolation distance between the surface and re-scanning planes. Using atomic force microscopy, we show that robust topographic changes can be imposed without altering the Young's modulus measured at the substrate surface by scanning at a depth of greater than ~6 µm. Transmission electron microscopy of hydrogel thin sections reveals changes to hydrogel porosity and density distribution within scanned regions, and that such changes to the hydrogel matrix are highly localized to regions of laser exposure. These results represent valuable new capabilities for deconvolving the effects of substrate dynamic physical attributes on the behavior of adherent cells.

In Situ Imprinting of Topographic Landscapes at the Cell-Substrate Interface.

In their native environments, adherent cells encounter dynamic topographical cues involved in promoting differentiation, orientation, and migration. Ideally, such processes would be amenable to study in cell culture using tools capable of imposing dynamic, arbitrary, and reversible topographic features without perturbing environmental conditions or causing chemical and/or structural disruptions to the substrate surface. To address this need, we report here development of an in vitro strategy for challenging cells with dynamic topographical experiences in which protein-based hydrogel substrate surfaces are modified in real time by positioning a pulsed, near-infrared laser focus within the hydrogel, promoting chemical cross-linking which results in local contraction of the protein matrix. Scanning the laser focus through arbitrary patterns directed by a dynamic reflective mask creates an internal contraction pattern that is projected onto the hydrogel surface as features such as rings, pegs, and grooves. By subjecting substrates to a sequence of scan patterns, we show that topographic features can be created, then eliminated or even reversed. Because laser-induced shrinkage can be confined to 3D voxels isolated from the cell-substrate interface, hydrogel modifications are made without damaging cells or disrupting the chemical or structural integrity of the surface.

Three-Dimensional Printing of Photoresponsive Biomaterials for Control of Bacterial Microenvironments.

Advances in microscopic three-dimensional (µ3D) printing provide a means to microfabricate an almost limitless range of arbitrary geometries, offering new opportunities to rapidly prototype complex architectures for microfluidic and cellular applications. Such 3D lithographic capabilities present a tantalizing prospect for engineering micromechanical components, for example, pumps and valves, for cellular environments composed of smart materials whose size, shape, permeability, stiffness, and other attributes might be modified in real time to precisely manipulate ultralow-volume samples. Unfortunately, most materials produced using µ3D printing are synthetic polymers that are inert to biologically tolerated chemical and light-based triggers and provide low compatibility as materials for cell culture and encapsulation applications. We previously demonstrated feasibility for µ3D printing environmentally sensitive, microstructured protein hydrogels that undergo volume changes in response to pH, ionic strength, and thermal triggers, cues that may be incompatible with sensitive chemical and biological systems. Here, we report the systematic investigation of photoillumination as a minimally invasive and remotely applied means to trigger morphological change in protein-based µ3D-printed smart materials. Detailed knowledge of material responsiveness is exploited to develop individually addressable "smart" valves that can be used to capture, "farm", and then dilute motile bacteria at specified times in multichamber picoliter edifices, capabilities that offer new opportunities for studying cell-cell interactions in ultralow-volume environments.

3D printing of microscopic bacterial communities.

Bacteria communicate via short-range physical and chemical signals, interactions known to mediate quorum sensing, sporulation, and other adaptive phenotypes. Although most in vitro studies examine bacterial properties averaged over large populations, the levels of key molecular determinants of bacterial fitness and pathogenicity (e.g., oxygen, quorum-sensing signals) may vary over micrometer scales within small, dense cellular aggregates believed to play key roles in disease transmission. A detailed understanding of how cell-cell interactions contribute to pathogenicity in natural, complex environments will require a new level of control in constructing more relevant cellular models for assessing bacterial phenotypes. Here, we describe a microscopic three-dimensional (3D) printing strategy that enables multiple populations of bacteria to be organized within essentially any 3D geometry, including adjacent, nested, and free-floating colonies. In this laser-based lithographic technique, microscopic containers are formed around selected bacteria suspended in gelatin via focal cross-linking of polypeptide molecules. After excess reagent is removed, trapped bacteria are localized within sealed cavities formed by the cross-linked gelatin, a highly porous material that supports rapid growth of fully enclosed cellular populations and readily transmits numerous biologically active species, including polypeptides, antibiotics, and quorum-sensing signals. Using this approach, we show that a picoliter-volume aggregate of Staphylococcus aureus can display substantial resistance to ß-lactam antibiotics by enclosure within a shell composed of Pseudomonas aeruginosa.

Multi-focal multiphoton lithography.

Multiphoton lithography (MPL) provides unparalleled capabilities for creating high-resolution, three-dimensional (3D) materials from a broad spectrum of building blocks and with few limitations on geometry, qualities that have been key to the design of chemically, mechanically, and biologically functional microforms. Unfortunately, the reliance of MPL on laser scanning limits the speed at which fabrication can be performed, making it impractical in many instances to produce large-scale, high-resolution objects such as complex micromachines, 3D microfluidics, etc. Previously, others have demonstrated the possibility of using multiple laser foci to simultaneously perform MPL at numerous sites in parallel, but use of a stage-scanning system to specify fabrication coordinates resulted in the production of identical features at each focal position. As a more general solution to the bottleneck problem, we demonstrate here the feasibility for performing multi-focal MPL using a dynamic mask to differentially modulate foci, an approach that enables each fabrication site to create independent (uncorrelated) features within a larger, integrated microform. In this proof-of-concept study, two simultaneously scanned foci produced the expected two-fold decrease in fabrication time, and this approach could be readily extended to many scanning foci by using a more powerful laser. Finally, we show that use of multiple foci in MPL can be exploited to assign heterogeneous properties (such as differential swelling) to micromaterials at distinct positions within a fabrication zone.

Building on the foundation of daring hypotheses: using the MKK4 metastasis suppressor to develop models of dormancy and metastatic colonization.

The identification of a novel metastasis suppressor function for the MAP Kinase Kinase 4 protein established a role for the stress-activated kinases in regulating the growth of disseminated cancer cells. In this review, we describe MKK4's biological mechanism of action and how this information is being used to guide the development of new models to study cancer cell dormancy and metastatic colonization. Specifically, we describe the novel application of microvolume structures, which can be modified to represent characteristics similar to those that cancer cells experience at metastatic sites. Although MKK4 is currently one of many known metastasis suppressors, this field of research started with a single daring hypothesis, which revolutionized our understanding of metastasis, and opened up new areas of exploration for basic research. The combination of our increasing knowledge of metastasis suppressors and such novel technologies provide hope for possible clinical interventions to prevent suffering from the burden of metastatic disease.

Multiphoton lithography using a high-repetition rate microchip laser.

Multiphoton lithography (MPL) provides a means to create prototype, three-dimensional (3D) materials for numerous applications in analysis and cell biology. A major impediment to the broad adoption of MPL in research laboratories is its reliance on high peak-power light sources, a requirement that typically has been met using expensive femtosecond titanium:sapphire lasers. Development of affordable microchip laser sources has the potential to substantially extend the reach of MPL, but previous lasers have provided relatively low pulse repetition rates (low kilohertz range), thereby limiting the rate at which microforms could be produced using this direct-write approach. In this report, we examine the MPL capabilities of a new, high-repetition-rate (36.6 kHz) microchip Nd:YAG laser. We show that this laser enables an approximate 4-fold decrease in fabrication times for protein-based microforms relative to the existing state-of-the-art microchip source and demonstrate its utility for creating complex 3D microarchitectures.

Microsecond analysis of transient molecules using bi-directional capillary electrophoresis.

We demonstrate the feasibility for minimizing electrophoretic analysis times of transient chemical species by inducing nascent, oppositely charged photochemical products to migrate in opposite directions from their point of creation. In this approach, separate probe sites are positioned within an electrophoretic channel both upfield and downfield from a photoreaction site formed by high-numerical-aperture optics, with positively charged (and in some cases neutral) components migrating toward one probe site and negatively charged species migrating in the opposite direction, toward the second probe site. As a proof-of-concept, fluorescent photoproducts of the hydroxyindoles, 5-hydroxytryptamine (serotonin), 5-hydroxytrptophan, and 5-hydroxyindole-2-carboxylic acid, are formed within a geometrically modified capillary and are transported electrophoretically and electroosmotically to probe sites several micrometers away. Although it is possible to detect all components in a single channel, or to use a two-channel imaging approach to independently detect positive and negative components, we have found the most rapid analysis approach involves a protocol in which laser light is alternately directed to opposing probe sites at high frequency (1 kHz), a strategy that allows positive and negative species to be detected with no cross-talk, even when components have overlapping detection times. Fluorescence-signal-averaging is performed on each temporal channel via summation of the two sequences of interdigitized electrophoretic traces. This approach allows photoproducts to be detected free from interferences from oppositely charged species, enabling positive and negative species in a mixture to be analyzed electrophoretically in ca. 6 micros, a period several-fold faster than was previously feasible using unidirectional electrophoresis.

Modular fluorescent benzobis(imidazolium) salts: syntheses, photophysical analyses, and applications.

A series of benzobis(imidazolium) (BBI) salts has been prepared and studied as a new class of versatile fluorescent materials. Using a high yielding, modular synthetic strategy, BBI salts with a range of functionality poised for investigating fundamental and applications-oriented characteristics, including emission wavelength tunability, solvatochromism, red-edge excitation, chemical stability, multiphoton excitation, and protein conjugation, were prepared in overall yields of 40-97%. Through structural variation, the BBIs exhibited lambda(em) ranging between 329 and 561 nm while displaying phi(f)s up to 0.91. In addition, the emission characteristics of these salts were found to exhibit strong solvent dependencies with Stokes shifts ranging from 4570 to 13 793 cm(-1), depending on the nature of the BBI core. Although red-edge effects for BBI salts with Br and BF4 counterions were found to be similar, unique characteristics were displayed by an analogue with MeSO4 anions. The stability of an amphiphilic BBI was quantified in aqueous solutions of varying pH, and >85% of the emission intensity was retained after 2 h at pH 3-9. Through multiphoton excitation experiments in aqueous solutions, a BBI salt was found to exhibit three-photon fluorescence action cross sections similar to serotonin. The application of BBI salts as fluorescent protein tags was demonstrated by conjugating bovine serum albumin to a maleimide-functionalized derivative.