Small, mobile FcepsilonRI receptor aggregates are signaling competent.

Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

In-fiber Mach-Zehnder interferometer for gas refractive index measurements based on a hollow-core photonic crystal fiber.

We describe an in-fiber interferometer based on a gas-filled hollow-core photonic crystal fiber. Expressions for the sensitivity, figure of merit and refractive index resolution are derived, and values are experimentally measured and theoretically validated using mode field calculations. The refractive indices of nine monoatomic and molecular gases are measured with a resolution of δns < 10-6.

Quantification of different water species in acetone using a NIR-triple-wavelength fiber laser.

A fiber laser using a thulium-doped ZBLAN gain medium was used to generate laser radiation simultaneously at 1461, 1505 and 1874 nm, with > 5 mW output power at each of the wavelengths. The laser was used to quantify the near-infrared absorption of liquid water in acetone. Additionally, near-infrared spectra were recorded using a broad band source and were interpreted using parallel factor (PARAFAC) analysis to rationalize the concentration-dependent peak shifts.

Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor FcϵRI of mast cells.

Current models propose that the plasma membrane of animal cells is composed of heterogeneous and dynamic microdomains known variously as cytoskeletal corrals, lipid rafts and protein islands. Much of the experimental evidence for these membrane compartments is indirect. Recently, live cell single particle tracking studies using quantum dot-labeled IgE bound to its high affinity receptor FcϵRI, provided direct evidence for the confinement of receptors within micrometer-scale cytoskeletal corrals. In this study, we show that an innovative time-series analysis of single particle tracking data for the high affinity IgE receptor, FcϵRI, on mast cells provides substantial quantitative information about the submicrometer organization of the membrane. The analysis focuses on the probability distribution function of the lengths of the jumps in the positions of the quantum dots labeling individual IgE FcϵRI complexes between frames in movies of their motion. Our results demonstrate the presence, within the micrometer-scale cytoskeletal corrals, of smaller subdomains that provide an additional level of receptor confinement. There is no characteristic size for these subdomains; their size varies smoothly from a few tens of nanometers to a over a hundred nanometers. In QD-IGE labeled unstimulated cells, jumps of less than 70 nm predominate over longer jumps. Addition of multivalent antigen to crosslink the QD-IgE-FcϵRI complexes causes a rapid slowing of receptor motion followed by a long tail of mostly jumps less than 70 nm. The reduced receptor mobility likely reflects both the membrane heterogeneity revealed by the confined motion of the monomeric receptor complexes and the antigen-induced cross linking of these complexes into dimers and higher oligomers. In both cases, the probability distribution of the jump lengths is well fit, from 10 nm to over 100 nm, by a novel power law. The fit for short jumps suggests that the motion of the quantum dots can be modeled as diffusion in a fractal space of dimension less than two.

Hyperspectral confocal fluorescence imaging: exploring alternative multivariate curve resolution approaches.

Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.

Water-soluble germanium(0) nanocrystals: cell recognition and near-infrared photothermal conversion properties.

Surfactant-passivated germanium nanocrystals (Ge(0) NCs) 3-5 nm in diameter were synthesized and encapsulated with functionalized phospholipids to yield water-soluble Ge(0) NCs. Upon encapsulation, the NCs retained their cubic crystalline phase and displayed good resistance to oxidation, as determined by transmission electron microscopy and X-ray photoelectron spectroscopy. As a test of their cell compatibility, the ability of carboxyfluorescein (CF)-labeled dinitrophenyl (DNP)-functionalized Ge(0) NCs to crosslink dinitrophenol-specific immunoglobulin E antibodies on the surface of mast cells (RBL-2H3) was examined in vitro. Treatment with a multivalent DNP antigen (i.e., DNP-Ge(0) NCs or CF-DNP-Ge(0) NCs) caused crosslinking of FcepsilonRI receptors and cellular responses, which were evaluated with morphological and colorimetric assays and live-cell fluorescence microscopy. Incubation of RBL-2H3 cells with Ge(0) NCs for approximately 24 h gave less than a 2 % increase in cell death as compared to DNP-functionalized bovine serum albumin. When irradiated with near-infrared (NIR) radiation (lambda(exc)=770 nm, 1.1 W cm(-2)) from a continuous-wave Ti:sapphire laser, the bulk-solution temperature of a toluene solution containing 20 mg mL(-1) Ge(0) NCs increased by approximately 35 degrees C within 5 min. Phospholipid-encapsulated water-soluble Ge(0) NCs at concentrations of 1.0 mg mL(-1) also displayed stable photothermal behavior under repetitive and prolonged NIR laser exposures in water, to yield a temperature increase of approximately 20 degrees C within 5 min (lambda(exc)=770 nm, 0.9 W cm(-2)). The photothermal efficiency of water-soluble Ge(0) NCs compares favorably with a recent report for Au nanoshells.

Synthesizing biofunctionalized nanoparticles to image cell signaling pathways.

This minireview outlines the synthetic efforts, from our research group, to produce nanomaterials for use as imaging agents to study cell signaling pathways. An overview of our approach to the synthesis and biofunctionalization of metal, semiconductor, and ceramic nanomaterials is presented. The probes investigated include coinage metals, Cd-based, Ge(o), naturally occurring fluorescent (NOF) minerals, and Ln-based nanoparticles which were synthesized from novel metal alkoxide, amide, and alkyl precursors. We illustrate the applications of some of these materials as imaging probes to detect signaling pathway components and cellular responses to signals (apoptosis and degranulation) in inflammatory and cancer cells.

Actin restricts FcepsilonRI diffusion and facilitates antigen-induced receptor immobilization.

The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcepsilonRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganization of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time-dependent. Multiple FcepsilonRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcepsilonRI signalling is activated by crosslinking with multivalent antigen. We show that receptors become immobilized within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilization kinetics and increased diffusion of crosslinked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration and response to ligand binding.

Speciation in the AlCl3/SO2Cl2 catholyte system.

The fundamental chemical behavior of the AlCl(3)/SO(2)Cl(2) catholyte system was investigated using (27)Al NMR spectroscopy, Raman spectroscopy, and single-crystal X-ray diffraction. Three major Al-containing species were found to be present in this catholyte system, where the ratio of each was dependent upon aging time, concentration, and/or storage temperature. The first species was identified as [Cl(2)Al(mu-Cl)](2) in equilibrium with AlCl(3). The second species results from the decomposition of SO(2)Cl(2) which forms Cl(2)(g) and SO(2)(g). The SO(2)(g) is readily consumed in the presence of AlCl(3) to form the crystallographically characterized species [Cl(2)Al(mu-O(2)SCl)](2) (1). For 1, each Al is tetrahedrally (T(d)) bound by two terminal Cl and two mu-O ligands whereas, the S is three-coordinated by two mu-O ligands and one terminal Cl. The third molecular species also has T(d)-coordinated Al metal centers but with increased oxygen coordination. Over time it was noted that a precipitate formed from the catholyte solutions. Raman spectroscopic studies show that this gel or precipitate has a component that was consistent with thionyl chloride. We have proposed a polymerization scheme that accounts for the precipitate formation. Further NMR studies indicate that the precipitate is in equilibrium with the solution.

Structural variations of potassium aryloxides.

A series of potassium aryloxides (KOAr) were isolated from the reaction of a potassium amide (KN(SiMe(3))(2)) and the desired substituted phenoxide (oMP, 2-methyl; oPP, 2-iso-propyl; oBP, 2-tert-butyl; DMP, 2,6-di-methyl; DIP, 2,6-di-iso-propyl; DBP, 2,6-di-tert-butyl) in tetrahydrofuran (THF) or pyridine (py) as the following: [([K(mu(4)-oMP)(THF)][K(mu(3)-oMP)])(5)]( infinity ) (1), [[K(6)(eta(6),mu(3)-oMP)(4)(eta(6),mu(4)-oMP)(2)(py)(4)].[K(6)(eta(6),mu(3)-oMP)(6)(eta(6)-py)(4)]]( infinity ) (2), [K(mu(3)-oPP)](4)(THF)(3) (3), [K(4)(eta(6),mu(3)-oPP)(2)(mu(3)-oPP)(2)(py)(3)]( infinity ) (4), [K(mu(3)-oBP)(THF)](6) (5), [K(6)(eta(6),mu(3)-oBP)(2)(mu(3)-oBP)(4)(py)(4)]( infinity ) (6), [K(3)(eta(6),mu(3)-DMP)(2)(mu-DMP)(THF)]( infinity ) (7), [[K(eta(6),mu-DMP)(py)](2)]( infinity ) (8), [K(eta(6),mu-DIP)]( infinity ) (9), [K(eta(6),mu-DBP)]( infinity ) (10). Further exploration of the aryl interactions led to the investigation of the diphenylethoxide (DPE) derivative which was isolated as [K(mu(3)-DPE)(THF)](4) (11) or [K(mu(3)-DPE)(py)](4).py(2) (12) depending on the solvent used. In general, the less sterically demanding ligands (oMP, oPP, oBP, and DMP) were solvated polymeric species; however, increasing the steric bulk (DIP and DBP) led to unsolvated polymers and not discrete molecules. For most of this novel family of compounds, the K atoms were pi-bound to the aryl rings of the neighboring phenoxide derivatives to fill their coordination sites. The synthesss and characterization of these compounds are described in detail.