The Certification of the Mass fractions of PAH Metabolites (1-hydroxyphenanthrene, 1-hydroxypyrene, and 3-hydroxybenzo(a)pyrene) in Two Fish Bile Materials, BCR-720 (sedement-exposed flounder bile), BCR-721 (oil-exposed plaice bile).

Abstract not availableJRC.D-Institute for Reference Materials and Measurements (Geel

Circular spectropolarimetric sensing of chiral photosystems in decaying leaves

Circular polarization spectroscopy has proven to be an indispensable tool in photosynthesis research and (bio)-molecular research in general. Oxygenic photosystems typically display an asymmetric Cotton effect around the chlorophyll absorbance maximum with a signal $\leq 1 \%$. In vegetation, these signals are the direct result of the chirality of the supramolecular aggregates. The circular polarization is thus directly influenced by the composition and architecture of the photosynthetic macrodomains, and is thereby linked to photosynthetic functioning. Although ordinarily measured only on a molecular level, we have developed a new spectropolarimetric instrument, TreePol, that allows for both laboratory and in-the-field measurements. Through spectral multiplexing, TreePol is capable of fast measurements with a sensitivity of $\sim 1*10^{-4}$ and is therefore suitable of non-destructively probing the molecular architecture of whole plant leaves. We have measured the chiroptical evolution of \textit{Hedera helix} leaves for a period of 22 days. Spectrally resolved circular polarization measurements (450-900 nm) on whole leaves in transmission exhibit a strong decrease in the polarization signal over time after plucking, which we accredit to the deterioration of chiral macro-aggregates. Chlorophyll \textit{a} levels measured over the same period by means of UV-Vis absorption and fluorescence spectroscopy showed a much smaller decrease. With these results we are able to distinguish healthy from deteriorating leaves. Hereby we indicate the potency of circular polarization spectroscopy on whole and intact leaves as a nondestructive tool for structural and plant stress assessment. Additionally, we underline the establishment of circular polarization signals as remotely accessible means of detecting the presence of extraterrestrial life.Comment: 29 pages, 6 figure

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study we have measured the circular polarizance of several multicellular green, red and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.Comment: 25 pages, 9 figure

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases

Imaging linear and circular polarization features in leaves with complete Mueller matrix polarimetry

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of l-MTX and its enantiomeric impurity d-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10−7 M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of l-MTX. Furthermore, l-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples

Time-resolved Raman spectroscopy for non-invasive detection through non-transparent materials

A study was conducted to demonstrate the use of time-resolved Raman spectroscopy (TRRS) for non-invasive detection through non-transparent materials. TRRS techniques relied on short laser pulses and gated detection to make the discrimination, while deeper Raman photons arrived at the detector later and helped in the selective collection of these photons by delaying the opening of a short detector gate. The short gate width used in TRRS considerably reduced fluorescence and it was found that TRRS measurements using an intensified charge-coupled device (ICCD) device to create a gate of 250 ps, provided better selectivity over spatially offset Raman spectroscopy (SORS) in sub-surface measurements on a mm-cm depth scale. The ability to obtain chemical information through opaque, diffusely-scattering layers showed the potential for a number of fields, including forensics and security screening