Whispering gallery mode resonators in continuous flow: spectral assignments and sensing with monodisperse microspheres.

Whispering gallery mode resonator (WGMR) microspheres yield highly structured optical spectra that are extremely sensitive to their environment and are of intense interest for use in a variety of sensing applications. Many efforts to leverage the unique sensitivities of WGMRs have relied on stringent experimental requirements to correlate specific spectral shifts/changes to an analyte/stimulus such as (1) precise positional knowledge, (2) reference spectra for each microsphere, and (3) high mechanical stability. Consequently, these factors can hinder adequate mixing or incorporation of analytes and can create challenges for remote sensing. This work describes a continuous flow technique for measuring whispering gallery mode (WGM) spectra of dye-doped microspheres suspended in solution and an accompanying analysis scheme that can extract the local refractive index without a priori knowledge of the microsphere size and position and without a reference spectrum. This measurement technique and analysis scheme was shown to accurately measure the refractive index of a range of alcohol and saline solutions down to a few thousandths of a refractive index unit (RIU). Additionally, a spectral clustering algorithm was applied to the fit results of two batches of microspheres suspended in water and was able to accurately assign spectra back to either batch of microspheres.

Tyrosine-derived stimuli responsive, fluorescent amino acids.

A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and meta-phenylenevinylene (m-PPV) backbone have been synthesized and their optical properties were studied. These novel UAAs were derived from protected diiodo-l-tyrosine using palladium-catalyzed Heck couplings with a series of styrene analogs. Unlike the other fluorescent UAAs, whose emissions are restricted to a narrow range of wavelengths, these new amino acids display the emission peaks at broad range wavelengths (from 400-800 nm); including NIR with QY of 4% in HEPES buffer. The incorporation of both pyridine and phenol functional groups leads to distinct red, green, and blue (RGB) emission, in its basic, acidic and neutral states, respectively. More importantly, these amino acids showed reversible pH and redox response showing their promise as stimuli responsive fluorescent probes. To further demonstrate the utility of these UAAs in peptide synthesis, one of the amino acids was incorporated into a cell penetrating peptide (CPP) sequence through standard solid phase peptide synthesis. Resultant CPP was treated with two different cell lines and the internalization was monitored by confocal fluorescence microscopy.

c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response.

BACKGROUND: The pathogenic Yersinia species exhibit a primarily extracellular lifestyle through manipulation of host signaling pathways that regulate pro-inflammatory gene expression and cytokine release. To identify host genes that are targeted by Yersinia during the infection process, we performed an RNA interference (RNAi) screen based on recovery of host NF-κB-mediated gene activation in response to TNF-α stimulation upon Y. enterocolitica infection. RESULTS: We screened shRNAs against 782 genes in the human kinome and 26 heat shock genes, and identified 19 genes that exhibited ≥ 40% relative increase in NF-κB reporter gene activity. The identified genes function in multiple cellular processes including MAP and ERK signaling pathways, ion channel activity, and regulation of cell growth. Pre-treatment with small molecule inhibitors specific for the screen hits c-KIT and CKII recovered NF-κB gene activation and/or pro-inflammatory TNF-α cytokine release in multiple cell types, in response to either Y. enterocolitica or Y. pestis infection. CONCLUSIONS: We demonstrate that pathogenic Yersinia exploits c-KIT signaling in a T3SS-dependent manner to downregulate expression of transcription factors EGR1 and RelA/p65, and pro-inflammatory cytokines. This study is the first major functional genomics RNAi screen to elucidate virulence mechanisms of a pathogen that is primarily dependent on extracellular-directed immunomodulation of host signaling pathways for suppression of host immunity.

Characterization of chemical speciation in ultrathin uranium oxide layered films.

A unique approach to detect chemical speciation and distribution on nanometer-scale nuclear materials has been achieved by the combination of neutron reflectometry and shell-isolated surface-enhanced Raman spectroscopy. Both surface and underlying layers of the uranium oxide materials were determined with angstrom-level resolution. Our results reveal that the UO(x) film is composed of three sublayers: an ∼38 Å thick layer of U(3)O(8) formed along the UO(x)/substrate interface; the adjacent sublayer consists of an ∼900 Å thick single phase of α-UO(3), and the top layer is γ-UO(3) with a thickness of ∼115 Å.

Live Cells as Dynamic Laboratories: Time Lapse Raman Spectral Microscopy of Nanoparticles with Both IgE Targeting and pH-Sensing Functions.

This review captures the use of live cells as dynamic microlaboratories through implementation of labeled nanoparticles (nanosensors) that have both sensing and targeting functions. The addition of 2,4-ε-dinitrophenol-L-lysine (DNP) as a FcεRI targeting ligand and 4-mercaptopyridine (4-MPy) as a pH-sensing ligand enables spatial and temporal monitoring of FcεRI receptors and their pH environment within the endocytic pathway. To ensure reliability, the sensor is calibrated in vivo using the ionophore nigericin and standard buffer solutions to equilibrate the external [H(+)] concentration with that of the cell compartments. This review highlights the nanosensors, ability to traffic and respond to pH of receptor-bound nanosensors (1) at physiological temperature (37°C) versus room temperature (25°C), (2) after pharmacological treatment with bafilomycin, an H(+) ATPase pump inhibitor, or amiloride, an inhibitor of Na(+)/H(+) exchange, and (3) in response to both temperature and pharmacological treatment. Whole-cell, time lapse images are demonstrated to show the ability to transform live cells into dynamic laboratories to monitor temporal and spatial endosomal pH. The versatility of these probes shows promise for future applications relevant to intracellular trafficking and intelligent drug design.

Evidence for room temperature delignification of wood using hydrogen peroxide and manganese acetate as a catalyst.

Manganese acetate was found to catalyze the oxidative delignification of wood with hydrogen peroxide at room temperature. The delignification reaction was monitored by optical and Raman microscopy, and liquid chromatography/mass spectrometry. When exposed to H(2)O(2) and Mn(OAc)(3) in aqueous solution, poplar wood sections were converted into a fine powder-like material which consisted of individual wood cells within 4 days at room temperature and without agitation. Optical and Raman microscopy provided the spatial distribution of cellulose and lignin in the wood structure, and showed the preferential oxidation of lignin-rich middle lamellae. Raman spectra from the solid residue revealed a delignified and cellulose-rich material. Glucose yields following enzymatic hydrolysis were 20-40% higher in poplar sawdust pretreated with Mn(OAc)(3) for 2, 4, and 7 days at room temperature than those in sawdust exposed to water only for identical durations, suggesting the viability of this mild, inexpensive method for pretreatment of lignocellulosic biomass.

Reversible swelling of the cell wall of poplar biomass by ionic liquid at room temperature.

Time-resolved autofluorescence, Raman microspectroscopy, and scanning microprobe X-ray diffraction were combined in order to characterize lignocellulosic biomass from poplar trees and how it changes during treatment with the ionic liquid 1-n-ethyl-3-methylimidazolium acetate (EMIMAC) at room temperature. The EMIMAC penetrates the cell wall from the lumen, swelling the cell wall by about a factor of two towards the empty lumen. However, the middle lamella remains unchanged, preventing the cell wall from swelling outwards. During this swelling, most of the cellulose microfibrils are solubilized but chain migration is restricted and a small percentage of microfibrils persist. When the EMIMAC is expelled, the cellulose recrystallizes as microfibrils of cellulose I. There is little change in the relative chemical composition of the cell wall after treatment. The action of EMIMAC on the poplar cell wall at room temperature would therefore appear to be a reversible swelling and a reversible decrystallization of the cell wall.

SERS nanosensors that report pH of endocytic compartments during FcεRI transit.

Recently, the development of an IgE receptor (FcεRI)-targeted, pH-sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated by Nowak-Lovato and Rector (Appl Spectrosc 63:387-395, 2009). The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37 °C) versus room temperature (25 °C). As expected, we observed markedly slower progression of receptors through low-pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H(+) ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na(+)/H(+) exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15-20 min to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors.

Ionic liquid pretreatment of poplar wood at room temperature: swelling and incorporation of nanoparticles.

Lignocellulosic biomass offers economic and environmental advantages over corn starch for biofuels production. However, its fractionation currently requires energy-intensive pretreatments, due to the lignin chemical resistance and complex cell wall structure. Recently, ionic liquids have been used to dissolve biomass at high temperatures. In this study, thin sections of poplar wood were swollen by ionic liquid (1-ethyl-3-methylimidazolium acetate) pretreatment at room temperature. The samples contract when rinsed with deionized water. The controlled expansion and contraction of the wood structure can be used to incorporate enzymes and catalysts deep into the wood structure for improved pretreatments and accelerated cellulose hydrolysis. As a proof of concept, silver and gold nanoparticles of diameters ranging from 20 to 100 nm were incorporated at depths up to 4 mum. Confocal surface-enhanced Raman images at different depths show that a significant number of nanoparticles were incorporated into the pretreated sample, and they remained on the samples after rinsing. Quantitative X-ray fluorescence microanalyses indicate that the majority of nanoparticle incorporation occurs after an ionic liquid pretreatment of less than 1 h. In addition to improved pretreatments, the incorporation of materials and chemicals into wood and paper products enables isotope tracing, development of new sensing, and imaging capabilities.

Microfluidic flow-flash: method for investigating protein dynamics.

We report a new method, microfluidic flow-flash, for measuring protein reaction kinetics. The method couples a microscope imaging detection system with a microfluidic flow cell to reduce data acquisition times and sample consumption. This combination allows for the simultaneous collection of spectral and temporal information. The microfluidic flow cell design utilizes three-dimensional sheath flow to reduce sample dispersion and minimize sample consumption. The ability to alter the flow rates in the microfluidic flow cells allows a variety of time scales to be studied with submillisecond time resolution. The imaging detection system can be coupled with several spectroscopic probes including fluorescence and UV/visible absorbance spectroscopy. Here, we utilize the microfluidic flow-flash method to probe the kinetics of CO recombination or O2 binding to myoglobin after the laser-induced photolysis of CO from myoglobin by UV/visible absorbance spectral imaging.