Resonance CARS study of the structure of "green" and "red" chromophores within the red fluorescent protein DsRed

Vibrational spectra of red fluorescent protein DsRed have been studied for the first time by polarization-sensitive multiplex coherent anti-Stokes Raman scattering at two excitation wavelengths, 545 and 583 nm, in resonance with the absorption bands of the immature "green" and mature "red" protein chromophores. Overall vibrational patterns of both DsRed chromophores were found to be similar to each other and to differ from that of S65T-GFP at pH8. The combined analysis of our CARS data and published structural information suggest that both "green" and "red" DsRed species possess an extended chromophore structure. Consequently, our data suggest that pi-bonding system extension during isomerization around the cis peptide bond between Phe 65 and Gln 66 is a necessary but not sufficient step in DsRed chromophore maturation.     

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

Novel Determination of Organic Acids in Diesel and Motor Oil by Ion Chromatography

A robust analytical method is required for the determination of low-molecular weight organic acids, which are potential causes of refinery and internal combustion engine corrosion. The ion chromatographic method developed in this study allows the determination of acetic acid and formic acid in diesel oil mixtures with a motor oil volume fraction of up to 10%. The hydrophobic matrix is automatically removed in-line through a matrix elution step with organic solvent and nonaqueous anion-exchange analyte extraction. Acetic acid and formic acid, as the smallest and most acidic aliphatic naphthenic acids, were determined. Gradient anion-exchange chromatography on high-capacity columns in combination with suppressed conductivity detection allowed their selective and sensitive determination. Typical recovery values were from 82 to 107% for each acid in the matrices evaluated with reproducibility less than 5% for acid fortified samples.     

Surface-enhanced resonance Raman scattering of cytochrome P450-2D6 on coated silver hydrosols

Surface-enhanced resonance Raman scattering (SERRS) from dilute solutions (down to nanomolar concentrations) of human mono-oxygenase CYP2D6 is observed using aqueous dispersions of Ag nanoparticles (hydrosol) coated with self-assembled monolayers (SAMs) of mercaptoalkanoic acids of two different lengths. From a direct comparison with its resonance Raman spectrum in solution, CYP2D6 appears to fully retain its native structure upon adsorption on coated hydrosol through electrostatic interaction, while a structural change in the active site is observed when uncoated citrate-reduced hydrosol is used. Using SERRS on these biocompatible coated hydrosols, the effects of dextromethorphan on the enzyme's active site can be observed, demonstrating that CYP2D6 ability of binding substrates is preserved. Moreover, by tuning the wavelength of the exciting laser away from the main absorption band of the heme, the vibrational bands of the SAM coating are observed and analyzed to see how the presence of the protein affects the SAM structure.     

Separation of twenty-one naphthalene sulfonates by means of capillary electrophoresis

The production and use of naphthalene sulfonates can easily cause pollution of surface and other types of waters. In the present study, capillary electrophoresis in combination with UV absorption detection was used to separate 21 amino- and hydroxy-substituted naphthalene sulfonates which included multiple isomeric compounds. The influence of various parameters such as pH (which turned out to be extremely important), temperature of the surrounding air flow, and the use of buffer additives (micelles, cyclodextrins, organic modifiers) was studied. Complete separation of all analytes including the isomers, was achieved in two runs with a 50 mM boric acid/borate buffer, containing either 100 mM sodium dodecylsulfonate or 15% acetonitrile. The limits of detection obtained for the individual compounds typically were 20μgI⁻¹. River water samples spiked at this concentration level could be analysed using a simple three-step sample clean-up procedure.     

Laser spectroscopy in analytical chemistry:light on the next millennium

To date, lasers are widely accepted tools in analytical spectroscopy, involved in various stand-alone and hyphenated techniques. Furthermore, significant progress can be noted in this field. In this paper, first of all some laser characteristics are discussed. Subsequently, five selected topics are outlined to illustrate recent achievements and future developments:

1. Laser-induced fluorescence for detection in capillary electrophoresis, including the use of ultraviolet, continuous-wave lasers in combination with wavelength-resolved emission detection; the use of diode laser-induced fluorescence in the red region of the electromagnetic spectrum and the use of Ti:sapphire lasers for multiphoton-excited fluorescence detection.

2. Degenerate four-wave mixing for detection in liquid microseparation systems (based on the coherence of laser light).

3. Fluorescence line-narrowing spectroscopy for identification purposes, a cryogenic high-resolution molecular fluorescence technique with a high potential in environmental analysis.

4. Recent developments in Raman spectroscopy (including ultraviolet-Resonance Raman and hyphenation of liquid chromatography and Raman spectroscopy).

5. Use of lasers for sample introduction in inorganic analysis based on controlled material ablation.

The intensity of the 1602 cm−1 band in human cells is related to mitochondrial activity

We report a Raman band at 1602 cm⁻¹ in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium. Copyright © 2009 John Wiley & Sons, Ltd.     

Yellow persistent luminescence of Sr2SiO4:Eu2+,Dy3+

This paper reports the photoluminescence and afterglow of Sr₂SiO₄ doped with Eu²⁺ and Dy³⁺. Factors governing the formation of the monoclinic or orthorhombic phase of this ortho-silicate are described and the impact of the crystallographic modification on the luminescence and afterglow under UV and VUV excitation are discussed and insight in factors limiting the efficiency of this yellow afterglow material is given.     

Irreversible visual sensing of humidity using a cholesteric liquid crystal

Irreversible optical sensing of humidity by a doped cholesteric liquid crystal is achieved by using a thin film of nematic host E7 with a binaphthylorthosilicate ester as dopant (guest). The film changes its color from blue (to green to orange to red) to colorless when exposed to humidity as the dopant is hydrolyzed.