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