Eric T. Ritschdorff

3D printing of microscopic bacterial communities

Significance Bacteria within the human body commonly thrive within structured three-dimensional (3D) communities composed of multiple bacterial species. Organization of individuals and populations within bacterial aggregates is believed to play key roles in mediating community attributes, affecting, for example, the virulence of infections within the cystic fibrosis lung and oral cavity. To gain detailed insights into how geometry may influence pathogenicity, we describe a strategy for 3D printing bacterial communities in which physically distinct but chemically interactive populations of defined size, shape, and density can be organized into essentially any arrangement. Using this approach, we show that resistance of one pathogenic species to an antibiotic can enhance the resistance of a second species by virtue of their 3D relationship. 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 β-lactam antibiotics by enclosure within a shell composed of Pseudomonas aeruginosa.

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.

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.

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.

Functionalizing micro-3D-printed protein hydrogels for cell adhesion and patterning

The extracellular matrix has been shown to profoundly influence both cell morphology and numerous cellular processes - including adhesion, differentiation, and alignment - through a range of chemical, mechanical, and topographical features. In these studies, we investigate a versatile platform for functionalizing micro-3D-printed (μ-3DP) protein hydrogels via multiphoton excitation of benzophenone-biotin, a photoactivatable ligand capable of reacting with the hydrogel matrix, which is subsequently linked to a biotinylated cell-adhesive peptide through a NeutrAvidin® bridge. This functionalization strategy is potentially applicable to a broad range of hydrogel platforms, enabling biomolecules to be precisely patterned at specified locations within 3D materials. As proof-of-concept of this strategys utility, we demonstrate that chemical modifications can be made to μ-3DP protein hydrogels that enable Schwann cells to be patterned without altering the mechanical or topographical properties of the hydrogel to an extent that influences SC cell adhesion. The ability to independently control potential cellular cues within in vitro cellular microenvironments is essential to investigating decoupled effects of biomaterial properties on cell-matrix interactions. In addition, we demonstrate feasibility for generating arbitrary immobilized chemical gradient profiles, a result that opens important opportunities for understanding and controlling haptotactic behaviors, such as directed migration, that are key to various tissue regeneration applications.

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 MKK4s 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.

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.

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.

Laser fabrication of 3D microenvironments for small cellular populations

We present development of a lithographic strategy based on multiphoton-excited crosslinking of proteins and other biological molecules. Application of this approach to study cultured cells in physiologically relevant, 3D microenvironments will be discussed.