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 threedimensional (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 crosslinked 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.multiphoton lithography | microfabrication | antibiotic resistance | polymicrobial U ncovering relationships between structure and function remains a central goal of biology. At molecular dimensions, protein structure can be modified to systematically evaluate how conformation gives rise to ligand binding, catalysis, and other functional properties. On the far larger scale of ecological habitats, organization plays a similarly vital role in mediating "function," where the social behavior of organisms-including their reproduction rate, mobility, and involvement in cooperative and predatory relationships-depends on the spatial arrangement of the community. As with molecular function, a detailed understanding of how organization affects behavior of communities would benefit from technologies for creating variants of defined structure.Nowhere is the potential value of geometrical control more evident than in the study of microbial ecosystems. The burgeoning field of sociomicrobiology has revealed a richness in the mechanisms by which bacteria engage in cooperative and adversarial relationships, affecting nearby individuals through physical contact and modifications to the chemical composition of their shared microenvironment. Spatially dependent interactions can result from perturbations to the nutritional state of the local habitat, but also may be caused by release of di...

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.

Multiphoton lithography (MPL) is a highly versatile strategy for creating 3D microscale objects with complex geometrical arrangements, including nested boxes, interlocking blocks, and braided threads. Of the various chemistries used to produce solid forms in MPL, protein photocrosslinking has been of particular value in biological applications, yielding materials with high porosity, tunable elasticity, and a diverse set of chemical and biochemical properties. Unfortunately, the potential for object drift, and consequent distortion, during this direct‐write process has required that microforms be constructed in integral contact with an immobile surface, precluding fabrication of protein‐based objects that retain rotational and translational degrees of freedom. Here, the development of a high‐viscosity protein‐based reagent that can be used to fabricate complex 3D microstructures that are not adhered to a surface, including chains of Möbius strips, paddlewheels, and unconstrained (free‐floating) probes for bacterial motility, is reported.

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.

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