Ulrich Panne

Quantitative Imaging of Gold and Silver Nanoparticles in Single Eukaryotic Cells by Laser Ablation ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.

Methods for studying synaptosomal copper release

Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilsons, Alzheimers, and probably in prion protein diseases like Creutzfeld-Jakobs disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant Kd=0.43 +/- 0.07 microM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a Kd of 50 +/- 2.5 microM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).

Analysis of glass and glass melts during the vitrification process of fly and bottom ashes by laser-induced plasma spectroscopy. Part I : Normalization and plasma diagnostics

Abstract For laser-induced plasma spectroscopy (LIPS) analysis of the main components (Si, Al, and Ca) in glasses utilized for vitrification of ashes from waste incineration, a normalization procedure for line ratios is presented. Even in homogeneous glass samples, considerable pulse-to-pulse variations of the plasma electronic excitation temperature and electron density were observed because of changes in the material–laser interaction. A normalization procedure is outlined using Saha–Boltzmann equilibrium relationships to include the electronic excitation temperature and density in the calibration model. As a result of the normalization, the variation of the line ratios is reduced and linear calibrations for LIPS intensity ratios versus concentration ratios are achieved. For samples with high aluminum concentrations, the analysis was hampered by self-reversal effects.

Elemental Bioimaging in Kidney by LA–ICP–MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

On-line and in-situ detection of lead aerosols by plasma-spectroscopy and laser-excited atomic fluorescence spectroscopy

Abstract A set-up for on-line and size-segregated detection of lead in ultrafine aerosols was developed. Lead nitrate aerosols with particle diameters between 10 and 300 nm were generated by ultrasonic nebulization of aqueous Pb(NO3)2-solutions. A differential mobility particle sizer (DMPS) was used for size-resolved mass calibration. Either a miniaturized acetylene-air-flame or a laser-induced plasma (LIP) was employed for atomization. Lead was detected with a spectrograph and a gateable, intensified CCD-camera by atomic emission spectroscopy (AES) and laser excited atomic fluorescence (LEAF). Due to the lower sensitivity, for LIP-AES no size-resolved calibration was possible and for calibration with polydisperse aerosols a detection limit of 155 μg m−3 was found for lead. With LEAF and flame atomization, a linear calibration curve was obtained with on-line detection limits of 47 ng m−3 for lead. No dependence of the detection limit on the particle diameter was observed. For LEAF with a laser-induced plasma as atom source, a correlation between the detection limit and the particle diameter was found. The detection limit increased from 55 ng m−3 for a particle diameter of 48 nm to 130 ng m−3 for a particle diameter of 300 nm. The increasing detection limit with increasing particle diameter was probably due to the incomplete atomization of larger particles in the colder periphery of the plasma.

Multivariate classification of pigments and inks using combined Raman spectroscopy and LIBS

AbstractThe authenticity of objects and artifacts is often the focus of forensic analytic chemistry. In document fraud cases, the most important objective is to determine the origin of a particular ink. Here, we introduce a new approach which utilizes the combination of two analytical methods, namely Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS). The methods provide complementary information on both molecular and elemental composition of samples. The potential of this hyphenation of spectroscopic methods is demonstrated for ten blue and black ink samples on white paper. LIBS and Raman spectra from different inks were fused into a single data matrix, and the number of different groups of inks was determined through multivariate analysis, i.e., principal component analysis, soft independent modelling of class analogy, partial least-squares discriminant analysis, and support vector machine. In all cases, the results obtained with the combined LIBS and Raman spectra were found to be superior to those obtained with the individual Raman or LIBS data sets. FigureCombination of Raman spectroscopy and LIBS for improved classification of inks: score plot from PCA and experimental set-up

Process analysis of recycled thermoplasts from consumer electronics by laser-induced plasma spectroscopy.

An experimental setup for direct elemental analysis of recycled thermoplasts from consumer electronics by laser-induced plasma spectroscopy (LIPS, or laser-induced breakdown spectroscopy, LIBS) was realized. The combination of a echelle spectrograph, featuring a high resolution with a broad spectral coverage, with multivariate methods, such as PLS, PCR, and variable subset selection via a genetic algorithm, resulted in considerable improvements in selectivity and sensitivity for this complex matrix. With a normalization to carbon as internal standard, the limits of detection were in the ppm range. A preliminary pattern recognition study points to the possibility of polymer recognition via the line-rich echelle spectra. Several experiments at an extruder within a recycling plant demonstrated successfully the capability of LIPS for different kinds of routine on-line process analysis.

Chemical characterization and classification of pollen.

We report on the in situ characterization of tree pollen molecular composition based on Raman spectroscopy. Different from purification-based analysis, the nondestructive approach allows (i) to analyze various classes of molecules simultaneously at microscopic resolution and (ii) to acquire fingerprint-like chemical information that was used for the classification of pollen from different species. Hierarchical cluster analysis of spectra from fresh pollen samples of 15 species partly related at the genus level and family level indicates separation of species based on the complete Raman spectral signature and yields classification in accord with biological systematics. The results have implications for the further elucidation of pollen biochemistry and also for the development of chemistry-based online pollen identification methods.

Carbonate-coordinated metal complexes precede the formation of liquid amorphous mineral emulsions of divalent metal carbonates

During the mineralisation of metal carbonates MCO3 (M=Ca, Sr, Ba, Mn, Cd, Pb) liquid-like amorphous intermediates emerge. These intermediates that form via a liquid/liquid phase separation behave like a classical emulsion and are stabilized electrostatically. The occurrence of these intermediates is attributed to the formation of highly hydrated networks whose stability is mainly based on weak interactions and the variability of the metal-containing pre-critical clusters. Their existence and compositional freedom are evidenced by electrospray ionization mass spectrometry (ESI-MS). Liquid intermediates in non-classical crystallisation pathways seem to be more common than assumed.

Multiplexed Immunohistochemical Detection of Tumor Markers in Breast Cancer Tissue Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry

We optimized multiplexed immunohistochemistry (IHC) on breast cancer tissue. Up to 20 tumor markers are routinely evaluated for one patient, and thus, a common analysis results in a series of time consuming staining procedures. As an alternative, we used lanthanides for labeling of primary antibodies, which are applied in IHC. Laser ablation (LA) ICPMS was elaborated as a detection tool for multiplexed IHC of tissue sections. In this study, we optimized sample preparation steps and LA ICPMS parameters to achieve a sufficient signal-to-background ratio. The results prove the high selectivity of applied antibodies, which was sustained after labeling. Up to three tumor markers (Her 2, CK 7, and MUC 1) were detected simultaneously in a single multiplex analysis of a 5 μm thin breast cancer tissue at a laser spot size of 200 μm. Furthermore, the LA ICPMS results indicate a significantly higher expression level of MUC 1 compared to Her 2 and CK 7, which was not obvious from the conventionally stained tissue sections.