Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.Molecular two-photon excitation (TPE) was predicted by Goppert-Mayer in 1931 (1). Experimental observations of multiphoton processes awaited the invention of pulsed ruby lasers in 1960. Closely following the demonstration of secondharmonic generation (SHG), the first demonstration of nonlinear optics, two-photon absorption was utilized by Kaiser and Garrett to excite fluorescence emission in CaF2:Eu3+ (2).Three-photon excited fluorescence was observed and the three-photon absorption cross section for naphthalene crystals was estimated by Singh and Bradley in 1964 (3). Subsequently, multiphoton excitation and fluorescence has been used in molecular spectroscopy of various materials (4-8).A significant biological application of multiphoton excitation began with the invention of two-photon laser scanning microscopy (TPLSM) by Denk, Strickler, and Webb in 1990 (9). Originally devised for localized photochemical activation of caged biomolecules, TPE of photochemical polymer crosslinking also has provided a means for high-density threedimensional optical data storage at 1012 bits/cm3 (10).This article on multiphoton excitation is motivated by the emergence of TPLSM as a powerful new microscopy for three-dimensionally resolved fluorescence imaging of biological samples (11,12). The development of TPLSM has been propelled by rapid technological advances in laser scanning microscopy (LSM) (13), fluorescence probe synthesis, modelocked femtosecond lasers (14, 15), and computational threedimensional image reconstruction (16). Recently, threephoton excited fluorescence and its potential applications in imaging have also been reported for several fluorescent dyes (17)(18)(19)(20). Effective implementation of nonlinear laser microscopy, however, requires k...

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Tryptophan and serotonin were imaged with infrared illumination by three-photon excitation (3PE) of their native ultraviolet (UV) fluorescence. This technique, established by 3PE cross section measurements of tryptophan and the monoamines serotonin and dopamine, circumvents the limitations imposed by photodamage, scattering, and indiscriminate background encountered in other UV microscopies. Three-dimensionally resolved images are presented along with measurements of the serotonin concentration ( approximately 50 mM) and content (up to approximately 5 x 10(8) molecules) of individual secretory granules.

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...

We report a method for creating stimuli-responsive biomaterials in which scanning nonlinear excitation is used to photocrosslink proteins at submicrometer 3D coordinates. Proteins with differing hydration properties can be combined to achieve tunable volume changes that are rapid and reversible in response to changes in chemical environment. Protein matrices having arbitrary 3D topographies and definable density gradients over micrometer dimensions provide the ability to effect rapid (<1 sec) and precise mechanical manipulations by means of changes in hydrogel size and shape, and applicability of these materials to cell biology is shown through the fabrication of responsive bacterial cages.Escherichia coli ͉ multiphoton lithography ͉ nanobiotechnology ͉ protein hydrogels ͉ smart materials

We introduce a novel sensing mechanism for nitric oxide (NO) detection with a particular easily synthesized embodiment (NO(550)), which displays a rapid and linear response to NO with a red-shifted 1500-fold turn-on signal from a dark background. Excellent selectivity was observed against other reactive oxygen/nitrogen species, pH, and various substances that interfere with existing probes. NO(550) crosses cell membranes but not nuclear membranes and is suitable for both intra- and extracellular NO quantifications. Good cytocompatibility was found during in vitro studies with two different cell lines. The high specificity, dark background, facile synthesis, and low pH dependence make NO(550) a superior probe for NO detection when used as an imaging agent.

Microbes frequently live in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.Pseudomonas aeruginosa | scanning electrochemical microscopy | quorum sensing | 3D printing | pyocyanin B acterial populations are often found in nature as small, densely packed aggregates containing ∼10 1 -10 5 cells (1-5). These aggregates serve as building blocks for larger biofilm communities as well as a primary mode of transmission for pathogenic microbes (5-8). Similar to biofilm communities, aggregates develop microscale physical and chemical heterogeneity and display clinically relevant phenotypes, including enhanced antibiotic resistance (2,(8)(9)(10)(11)(12)(13)(14)(15)(16). Moreover, aggregate sizes containing as few as 10 3 bacteria have been shown to engage in quorum sensing (QS)-mediated behaviors (17-21). In its simplest form, QS is a communication strategy that allows bacteria to effectively monitor their population density through the secretion and sensing of extracellular signals (7,(22)(23)(24). When the population reaches a specific density, activation of the QS regulatory cascade results in enhanced transcription of a defined set of genes. These genes control distinct behaviors, including virulence, in the opportunistic pathogen Pseudomonas aeruginosa (25). In addition to displaying QS-mediated behaviors, bacterial aggregates have been shown to interact with neighboring aggregates both in vitro and in vivo (9,(26)(27)(28). Indeed, these interactions have a profound impact on virulence and are often mediated by small diffusible molecules (8-10, 22, 29-31).Despite the prevalence of aggregates in nature, understanding the mechanisms controlling their behavior and intera...

Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations.