A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically relevant context of the intact brain for which photons must traverse the cranium, skin, and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, this study describes optimal excitation and emission wavelengths for through-cranium imaging, and demonstrates that near-infrared emissive nanosensors can be photoexcited using a two-photon 1560 nm excitation source. Dopaminesensitive nanosensors can undergo two-photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. The two-photon absorption cross-section and quantum yield of dopamine nanosensors are further calculated, and a two-photon power law relationship for the nanosensor excitation process is confirmed. Finally, the improved image quality of the nanosensors embedded 2-mm-deep into a brain-mimetic tissue phantom is shown, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under two-photon excitation. The approach overcomes traditional limitations in deep-tissue fluorescence microscopy, and can enable neurotransmitter imaging in the biologically relevant milieu of the intact and living brain.

A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally-occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNT) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions, and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can selectively detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. This WGA protein nanosensor is characterized with near-infrared spectroscopy and microscopy to study protein-nanosensor interaction parameters. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin's conjugate sugars, suggesting the lectin protein selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs, and also demonstrate these assemblies can serve as optical nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.

Microbial redox activity offers a potentially transformative approach to the low-temperature synthesis of nanostructured inorganic materials. Diverse strains of the dissimilatory metal-reducing bacteria Shewanella are known to produce photoactive filamentous arsenic sulfide nanomaterials by reducing arsenate and thiosulfate in anaerobic culture conditions. Here we report in situ microscopic observations and measure the thermally activated (79 kJ mol(-1)) precipitation kinetics of high yield (504 mg per liter of culture, 82% of theoretical maximum) extracellular As2S3 nanofibers produced by Shewanella sp. strain ANA-3, and demonstrate their potential in functional devices by constructing field effect transistors (FETs) based on individual nanofibers. The use of strain ANA-3, which possesses both respiratory and detoxification arsenic reductases, resulted in significantly faster nanofiber synthesis than other strains previously tested, mutants of ANA-3 deficient in arsenic reduction, and when compared to abiotic arsenic sulfide precipitation from As(III) and S(2-). Detailed characterization by electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis and Tauc analysis of UV-vis spectrophotometry showed the biogenic precipitate to consist primarily of amorphous As2S3 nanofibers with an indirect optical band gap of 2.37 eV. X-ray diffraction also revealed the presence of crystalline As8S(9-x) minerals that, until recently, were thought to form only at higher temperatures and under hydrothermal conditions. The nanoscale FETs enabled a detailed characterization of the charge mobility (∼10(-5) cm(2) V(-1) s(-1)) and gating behavior of the heterogeneously doped nanofibers. These studies indicate that the biotransformation of metalloids and chalcogens by bacteria enables fast, efficient, sustainable synthesis of technologically relevant chalcogenides for potential electronic and optoelectronic applications.

Bacteria classified in species of the genus Leptothrix produce extracellular, microtubular, Fe-encrusted sheaths. The encrustation has been previously linked to bacterial Fe oxidases, which oxidize Fe(II) to Fe(III) and/or active groups of bacterial exopolymers within sheaths to attract and bind aqueous-phase inorganics. When L. cholodnii SP-6 cells were cultured in media amended with high Fe(II) concentrations, Fe(III) precipitates visibly formed immediately after addition of Fe(II) to the medium, suggesting prompt abiotic oxidation of Fe(II) to Fe(III). Intriguingly, these precipitates were deposited onto the sheath surface of bacterial cells as the population was actively growing. When Fe(III) was added to the medium, similar precipitates formed in the medium first and were abiotically deposited onto the sheath surfaces. The precipitates in the Fe(II) medium were composed of assemblies of globular, amorphous particles (ca. 50 nm diameter), while those in the Fe(III) medium were composed of large, aggregated particles (≥3 µm diameter) with a similar amorphous structure. These precipitates also adhered to cell-free sheaths. We thus concluded that direct abiotic deposition of Fe complexes onto the sheath surface occurs independently of cellular activity in liquid media containing Fe salts, although it remains unclear how this deposition is associated with the previously proposed mechanisms (oxidation enzyme- and/or active group of organic components-involved) of Fe encrustation of the Leptothrix sheaths.

Imaging the dynamic behavior of neuromodulatory neurotransmitters in the extracelluar space that arise from individual quantal release events would constitute a major advance in neurochemical imaging. Spatial and temporal resolution of these highly stochastic neuromodulatory events requires concurrent advances in the chemical development of optical nanosensors selective for neuromodulators in concert with advances in imaging methodologies to capture millisecond neurotransmitter release. Herein, we develop and implement a stochastic model to describe dopamine dynamics in the extracellular space (ECS) of the brain dorsal striatum to guide the design and implementation of fluorescent neurochemical probes that record neurotransmitter dynamics in the ECS. Our model is developed from first-principles and simulates release, diffusion, and reuptake of dopamine in a 3D simulation volume of striatal tissue. We find that in vivo imaging of neuromodulation requires simultaneous optimization of dopamine nanosensor reversibility and sensitivity: dopamine imaging in the striatum or nucleus accumbens requires nanosensors with an optimal dopamine dissociation constant (K) of 1 μM, whereas Ks above 10 μM are required for dopamine imaging in the prefrontal cortex. Furthermore, as a result of the probabilistic nature of dopamine terminal activity in the striatum, our model reveals that imaging frame rates of 20 Hz are optimal for recording temporally resolved dopamine release events. Our work provides a modeling platform to probe how complex neuromodulatory processes can be studied with fluorescent nanosensors and enables direct evaluation of nanosensor chemistry and imaging hardware parameters. Our stochastic model is generic for evaluating fluorescent neurotransmission probes, and is broadly applicable to the design of other neurotransmitter fluorophores and their optimization for implementation in vivo.