Ortsaufgelöster Nachweis von Elementspuren mittels Laserablation und Resonanzionisations-Massenspektrometrie und Untersuchungen zur Laserablations-Dynamik

Im Rahmen dieser Arbeit wurde eine neue Methode für einen empfindlichen und isotopenselektiven Elementnachweis entwickelt. Unter Einsatz von Laserablation geschieht der Probenaufschluß direkt und mit einer Ortsauflösung von unter 30 m. Hierzu wurde ein hochauflösendes MALDI-TOF-Massenspektrometer, welches üblicherweise für biochemische Fragestellungen eingesetzt wird, mit einem spektroskopischen Aufbau zur resonanten Ionisation von Elementgehalten modifiziert. Die Methode ist somit insbesondere für die Untersuchung von Elementspuren in Festkörperproben mit mikroskopischer Struktur konzipiert. Methodische Entwicklungsarbeiten wurden anhand des Elements Gadolinium durchgeführt. Durch die Verwendung gepulster Farbstofflaser stehen ausreichend hohe Laserfelder zur Verfügung, um unabhängig von Hyperfeinstruktur und Isotopieverschiebung Übergänge aller Isotope im Rahmen des Resonanzionisations-Verfahrens zu sättigen. Darauf konnte eine Isotopenverhältnisanalyse mit einer Genauigkeit im Prozentbereich verwirklicht werden. Verschiedene Anregungsleitern wurden untersucht, und mit elementspezifischen Resonanzüberhöhungen bis zu zwei Größenordnungen über dem nicht-resonant gebildeten Untergrund konnte eine Nachweiseffizienz von über 10-4 (entsprechend sub-fg/g-Niveau) erzielt werden. Dazu wurden Simulationsrechnungen zum atomaren Sättigungsverhalten in starken resonanten Laserfeldern durchgeführt. Erste Anwendungen des Laserablationsverfahrens waren Proben kosmologischer Herkunft. Der physikalische Prozeß der Laserablation bei Metallen wurde unter Hochvakuum-Bedingung systematisch in Abhängigkeit der Laserfluenz untersucht. In der ablatierten Plasmaphase erwies sich der Neutralanteil als besonders geeignet für geschwindigkeitsselektive Laserionisations-Messungen. Eine bimodale Struktur wurde beobachtet, bestehend aus einer thermischen und einer schockwellen-induzierten Komponente. Der ionische Anteil der ablatierten Dampfphase konnte über variable elektrische Feldpulse untersucht werden. Laserablation unter Atmosphärenbedingung wurde an einem beschichteten Messingtarget untersucht. Dabei wurde die Entstehung von permanenten Oberflächenstrukturen beobachtet, welche sich durch Nichtgleichgewichts-Prozesse in der Dampfphase erklären lassen.

Oligomerisation, localisation and interaction of the sensor histidine kinases DcuS and CitA in Escherichia coli

The two-component system DcuSR of Escherichia coli regulates gene expression of anaerobic fumarate respiration and aerobic C4-dicarboxylate uptake. C4-dicarboxylates and citrate are perceived by the periplasmic domain of the membrane-integral sensor histidine kinase DcuS. The signal is transduced across the membrane by phosphorylation of DcuS and of the response regulator DcuR, resulting in activation of DcuR and transcription of the target genes.rnIn this work, the oligomerisation of full-length DcuS was studied in vivo and in vitro. DcuS was genetically fused to derivatives of the green fluorescent protein (GFP), enabling fluorescence resonance energy transfer (FRET) measurements to detect protein-protein interactions in vivo. FRET measurements were also performed with purified His6-DcuS after labelling with fluorescent dyes and reconstitution into liposomes to study oligomerisation of DcuS in vitro. In vitro and in vivo fluorescence resonance energy transfer showed the presence of oligomeric DcuS in the membrane, which was independent of the presence of effector. Chemical crosslinking experiments allowed clear-cut evaluation of the oligomeric state of DcuS. The results showed that detergent-solubilised His6-DcuS was mainly monomeric and demonstrated the presence of tetrameric DcuS in proteoliposomes and in bacterial membranes.rnThe sensor histidine kinase CitA is part of the two-component system CitAB of E. coli, which is structurally related to DcuSR. CitAB regulates gene expression of citrate fermentation in response to external citrate. The sensor kinases DcuS and CitA were fused with an enhanced variant of the yellow fluorescent protein (YFP) and expressed in E. coli under the control of an arabinose-inducible promoter. The subcellular localisation of DcuS-YFP and CitA-YFP within the cell membrane was studied by means of confocal laser fluorescence microscopy. Both fusion proteins were found to accumulate at the cell poles. The polar accumulation was slightly increased in the presence of the stimulus fumarate or citrate, respectively, but independent of the expression level of the fusion proteins. Cell fractionation demonstrated that polar accumulation was not related to inclusion bodies formation. The degree of polar localisation of DcuS-YFP was similar to that of the well-characterised methyl-accepting chemotaxis proteins (MCPs), but independent of their presence. To enable further investigations on the function of the polar localisation of DcuS under physiological conditions, the sensor kinase was genetically fused to the flavin-based fluorescent protein Bs2 which shows fluorescence under aerobic and anaerobic conditions. The resulting dcuS-bs2 gene fusion was inserted into the chromosome of various E. coli strains.rnFurthermore, a protein-protein interaction between the related sensor histidine kinases DcuS and CitA, regulating common metabolic pathways, was detected via expression studies under anaerobic conditions in the presence of citrate and by in vivo FRET measurements.

Relationship between DIAGNOdent values and sealant penetration depth on occlusal fissures

The aim of this in vitro study was to evaluate the relationship between laser fluorescence values and sealant penetration depth on occlusal fissures. One hundred and sixty-six permanent molars were selected and divided into four groups, which were each treated using a different sealant (two clear and two opaque). The teeth were independently measured twice by two experienced dentists using two laser fluorescence devices-DIAGNOdent (LF and LFpen)-before and after sealing, and then thermoclycled. After measuring, the teeth were histologically prepared and assessed for caries extension. Digital photographs of the cut sealed sites were assessed, and the sealant penetration depth was measured. All 166 sites were measured by one of the examiners taking as limits the outer and inner surface of the sealant into the fissure. For each device (LF and LFpen) and each group, the difference between the values at baseline and after sealing was plotted against the sealant penetration depth and scatter plots were provided. It could be observed that most of the points were concentrated around the zero line, for both LF and LFpen in the four groups. In conclusion, there is no relation between changes in DIAGNOdent values and increasing of depth sealant penetration within the occlusal fissures.

Treating open carious lesions in anxious children with ozone. A prospective controlled clinical study

PURPOSE: To determine whether the treatment of dental caries with ozone was possible in apprehensive children and to ascertain whether ozone reverses caries in open single-surface lesions. Further, the influence of ozone on laser fluorescence was investigated. METHODS: 82 lesions in 28 children with at least two open single-surface lesions were assessed. The children were anxious and were judged by the referring dentist as non-treatable. For each test lesion, which was treated with ozone, a control lesion was left without ozone treatment. Hardness and laser fluorescence values were assessed and the changes for hardness and laser fluorescence values in the test lesion were compared with the values in the control lesion after 2, 4, 6, and 8 months. RESULTS: 94 percent of the children were treatable and 93% lost their dental anxiety. The hardness values improved significantly in the ozone-treated test lesions after 4, 6, and 8 months (P< 0.05) compared with baseline while the control lesions had no significant change in hardness at any recall interval. Comparing the differences between test and control teeth over time, the laser fluorescence values improved, however the improvement was not statistically significant (P> 0.05). The use of ozone resulted in an average reduction of 13% of the laser fluorescence values immediately after the ozone treatment.

Action of food preservatives on 14-days dental biofilm formation, biofilm vitality and biofilm-derived enamel demineralisation in situ

AIMS The aims of this double-blind, controlled, crossover study were to assess the influence of food preservatives on in situ dental biofilm growth and vitality, and to evaluate their influence on the ability of dental biofilm to demineralize underlying enamel over a period of 14 days. MATERIALS AND METHODS Twenty volunteers wore appliances with six specimens each of bovine enamel to build up intra-oral biofilms. During four test cycles of 14 days, the subjects had to place the appliance in one of the assigned controls or active solutions twice a day for a minute: negative control 0.9 % saline, 0.1 % benzoate (BA), 0.1 % sorbate (SA) and 0.2 % chlorhexidine (CHX positive control). After 14 days, the biofilms on two of the slabs were stained to visualize vital and dead bacteria to assess biofilm thickness (BT) and bacterial vitality (BV). Further, slabs were taken to determine mineral loss (ML), by quantitative light-induced laser fluorescence (QLF) and transversal microradiography (TMR), moreover the lesion depths (LD). RESULTS Nineteen subjects completed all test cycles. Use of SA, BA and CHX resulted in a significantly reduced BV compared to NaCl (p < 0.001). Only CHX exerted a statistically significant retardation in BT as compared to saline. Differences between SA and BA were not significant (p > 0.05) for both parameters. TMR analysis revealed the highest LD values in the NaCl group (43.6 ± 44.2 μm) and the lowest with CHX (11.7 ± 39.4 μm), while SA (22.9 ± 45.2 μm) and BA (21.4 ± 38.5 μm) lay in between. Similarly for ML, the highest mean values of 128.1 ± 207.3 vol% μm were assessed for NaCl, the lowest for CHX (-16.8 ± 284.2 vol% μm), while SA and BA led to values of 83.2 ± 150.9 and 98.4 ± 191.2 vol% μm, respectively. With QLF for both controls, NaCl (-33.8 ± 101.3 mm(2) %) and CHX (-16.9 ± 69.9 mm(2) %), negative values were recorded reflecting a diminution of fluorescence, while positive values were found with SA (33.9 ± 158.2 mm(2) %) and BA (24.8 ± 118.0 mm(2) %) depicting a fluorescence gain. These differences were non-significant (p > 0.05). CONCLUSION The biofilm model permited the assessment of undisturbed oral biofilm formation influenced by antibacterial components under clinical conditions for a period of 14 days. An effect of BA and SA on the demineralization of enamel could be demonstrated by TMR and QLF, but these new findings have to be seen as a trend. As part of our daily diet, these preservatives exert an impact on the metabolism of the dental biofilm, and therefore may even influence demineralization processes of the underlying dental enamel in situ.

Diagnostic performance of a new red light LED device for approximal caries detection

The aim of this study was to test a newly developed LED-based fluorescence device for approximal caries detection in vitro. We assembled 120 extracted molars without frank cavitations or fillings pairwise in order to create contact areas. The teeth were independently assessed by two examiners using visual caries detection (International Caries Detection and Assessment System, ICDAS), bitewing radiography (BW), laser fluorescence (LFpen), and LED fluorescence (Midwest Caries I.D., MW). The measurements were repeated at least 1 week later. The diagnostic performance was calculated with Bayesian analyses. Post-test probabilities were calculated in order to judge the diagnostic performance of combined methods. Reliability analyses were performed using kappa statistics for nominal data and intraclass correlation (ICC) for absolute data. Histology served as the gold standard. Sensitivities/specificities at the enamel threshold were 0.33/0.84 for ICDAS, 0.23/0.86 for BW, 0.47/0.78 for LFpen, and 0.32/0.87 for MW. Sensitivities/specificities at the dentine threshold were 0.04/0.89 for ICDAS, 0.27/0.94 for BW, 0.39/0.84 for LFpen, and 0.07/0.96 for MW. Reliability data were fair to moderate for MW and good for BW and LFpen. The combination of ICDAS and radiography yielded the best diagnostic performance (post-test probability of 0.73 at the dentine threshold). The newly developed LED device is not able to be recommended for approximal caries detection. There might be too much signal loss during signal transduction from the occlusal aspect to the proximal lesion site and the reverse.

Major, trace and rare earth element concentration of ODP Site 210-1277 basalts, Newfoundland margin

Drilling of the distal Newfoundland margin at Ocean Drilling Program Site 1277 recovered part of the transition between exhumed sub-continental mantle lithosphere and normal mid-ocean-ridge basalt (N-MORB) volcanism perhaps related to the initiation of seafloor spreading, which may have occurred near the Aptian/Albian boundary, coincident with the final separation of subcontinental mantle lithosphere. Subcontinental mantle lithosphere was recovered near the crest of a basement high, the Mauzy Ridge. This ridge lies near magnetic Anomaly M1 and is inferred to be of Barremian age. The recovered section is dominated by serpentinized spinel harzburgite, with subordinate dunite and minor gabbroic intrusives, and it includes inferred high-temperature ductile shear zones. The serpentinite is capped by foliated gabbro cataclasite that is interpreted as the product of a major seafloor extensional detachment. The serpentinized harzburgite beneath is highly depleted subcontinental mantle lithosphere that was exhumed to create new seafloor within the ocean-continent transition zone. After inferred removal of overlying brittle crust, the detachment was eroded, producing multiple mass flows that were dominated by clasts of serpentinite and gabbro in a lithoclastic and calcareous matrix. Basaltic lavas were erupted spasmodically, mainly as sheet flows, with subordinate lava breccia, hyaloclastite, and possible pillow lava. The sedimentary-volcanic succession and the exhumed mantle lithosphere experienced later high-angle extensional fracturing and probably faulting. Extensional fissures opened incrementally and were filled with silt-sized carbonate, basalt-derived clastic sediment, and hyaloclastite, forming neptunian dykes and geopetal structures. Chemical analysis of representative basalts for major elements and trace elements were made using a high-precision, high-accuracy X-ray fluorescence method (utilizing increased count times) and by whole-rock inductively coupled plasma-mass spectrometry that yielded additional evidence for rare earth elements. The analyses indicate N-MORB to slightly enriched compositions. The MORB was produced by relatively high degree melting of a fertile mantle source that differed strongly from the cored serpentinized peridotites. The basalts exhibit a distinct negative Nb anomaly on MORB-normalized plots that can be explained by prior extraction of melt from upper mantle that had previously been affected by subduction, possibly during closure of the Iapetus or Rheic oceans. In the proposed interpretation, mantle lithosphere was exhumed to the seafloor and experienced mass wasting to form serpentinite-rich mass flows. The interbedded MORB records the beginning of a transition to "normal" seafloor spreading. This interpretation takes into account drilling results from the Iberia-Galicia margin and the Jurassic Alps-Apennines.

Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na+], [Cl−], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1–0.5 μl of fluid accumulated in spherical droplets at gland orifices in ≈3–5 min. In gland fluid from normal human airways, [Na+] was 94 ± 8 mM, [Cl−] was 92 ± 12 mM, and pH was 6.97 ± 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 ± 0.3-fold greater than that of saline. Neither [Na+] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by ≈2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na+], [Cl−], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.

Cystic fibrosis transmembrane conductance regulator activation stimulates endosome fusion in vivo.

Previous studies have suggested a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of intracellular vesicular trafficking. A quantitative fluorescence method was used to test the hypothesis that CFTR expression and activation affects endosome-endosome fusion in intact cells. Endosomes from CFTR-expressing and control (vector-transfected) Swiss 3T3 fibroblasts were labeled by internalization with 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene (Bodipy)-avidin, a fluid-phase marker whose fluorescence increases approximately 8-fold upon biotin binding. Cells were washed, chased, and then labeled with biotin-albumin or biotin-transferrin. The fraction of Bodipy-avidin-labeled endosomes that fused with biotin-containing endosomes (f(fusion)) was quantified by ratio imaging microfluorimetry. Endosome fusion in unstimulated CFTR-expressing cells was similar to that in control cells. However, in CFTR-expressing cells activated by forskolin, ffusion was increased by 1.30 +/- 0.18- and 2.65 +/- 0.17-fold for a 0 and 10 min chase time between avidin and biotin-albumin pulses; f(fusion) also increased (1.32 +/- 0.11-fold) when biotin-transferrin replaced biotin-albumin. The stimulation of endosome fusion was not due to differences in rates of endocytosis or endosomal acidification. Endosome fusion was not stimulated by forskolin in Cl--depleted CFTR-expressing cells, suggesting that the increase in endosome fusion is due to the CFTR chloride channel activity. These results provide evidence that CFTR is involved in the regulation of endosome fusion and, thus, a possible basis for the cellular defects associated with cystic fibrosis.

Magnetophotoresistance in Pr 1–x CaxMnO3 Thin Films

In this work, a colossal magnetoresistive (CMR) Pr1−xCaxMnO3 (PCMO) man- ganite thin films and polycrystalline samples were studied with the main focus on the properties of the insulator to metal Mott phase transition. The polycrystalline PCMO samples were fabricated with the solid state reaction method. The polycrystalline sam- ples were further processed into the epitaxial thin films with the pulsed laser deposition method (PLD). The structural and magnetic properties of the samples were systemat- ically investigated and the thin films samples were further investigated with magneto- transport measurement where the thin films response to illumination was also studied. After the successful synthesis of polycrystalline PCMO samples with varying x = between 0.0 - 0.5, the magnetic characterization of the samples showed rich magnetic properties having the signatures of the magnetic phase coexistence of antiferromag- netic (AFM) and ferromagnetic (FM) ordering and cluster glass behaviour. With the increased doping concentration from x = 0.3 to 0.5, the AFM charge-order (CO) phase started to form up being strongest on the sample x = 0.5. This AFM CO phase could be melted with the high external magnetic field at temperatures below Neel’s tempera- ture TN inducing an irreversible first order metamagnetic AFM to FM phase transition. The strength of the AFM CO phase decreased with decreasing Ca concentration and increasing temperature. The polycrystalline PCMO samples with Ca concentrations of 0.3 - 0.5, showing metamagnetic behaviour, were selected for the fabrication of the thin film samples. The films were grown using two different in situ oxygen treatment temperatures at 500 ◦C and 700 ◦C in the PLD system. The films with x = 0.4 and 0.5 showed weaker AFM CO phase with greatly reduced melting fields when compared to polycrystalline samples. Also, the robustness of the AFM CO phase was further decreased in thin films with the lower oxygen treatment temperature of 500 ◦C. The magneto-transport measurements made on the thin films showed that the melting of AFM CO phase was connected to CMR effect where the increasing magnetic field induced an insulator to metal phase transition, which reduces the resistivity of the film around nine orders of magnitude. The use of illumination during the magneto-transport measurements showed a vari- ety of intriguing phenomena including magnetophotoresistance. The illumination had a huge effect to the insulator to metal transition (IMT) reducing the transition magnetic field significantly. Moreover, by magnetically biasing the thin films with the constant external magnetic field, the IMT could be induced by switching on the illumination.

Fetal-maternal interface impedance parallels local NADPH oxidase related superoxide production

Blood flow assessment employing Doppler techniques is a useful procedure in pregnancy evaluation, as it may predict pregnancy disorders coursing with increased uterine vascular impedance, as pre-eclampsia. While the local causes are unknown, emphasis has been put on reactive oxygen species (ROS) excessive production. As NADPH oxidase (NOX) is a ROS generator, it is hypothesized that combining Doppler assessment with NOX activity might provide useful knowledge on placental bed disorders underlying mechanisms. A prospective longitudinal study was performed in 19 normal course, singleton pregnancies. Fetal aortic isthmus (AoI) and maternal uterine arteries (UtA) pulsatility index (PI) were recorded at two time points: 20-22 and 40-41 weeks, just before elective Cesarean section. In addition, placenta and placental bed biopsies were performed immediately after fetal extraction. NOX activity was evaluated using a dihydroethidium-based fluorescence method and associations to PI values were studied with Spearman correlations. A clustering of pregnancies coursing with higher and lower PI values was shown, which correlated strongly with placental bed NOX activity, but less consistently with placental tissue. The study provides evidence favoring that placental bed NOX activity parallels UtA PI enhancement and suggests that an excess in oxidation underlies the development of pregnancy disorders coursing with enhanced UtA impedance.

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.

Autonomous extraction of optimal flame fronts in OH planar laser-induced fluorescence images.

The location of a flame front is often taken as the point of maximum OH gradient. Planar laser-induced fluorescence of OH can be used to obtain the flame front by extracting the points of maximum gradient. This operation is typically performed using an edge detection algorithm. The choice of operating parameters a priori poses significant problems of robustness when handling images with a range of signal-to-noise ratios. A statistical method of parameter selection originating in the image processing literature is detailed, and its merit for this application is demonstrated. A reduced search space method is proposed to decrease computational cost and render the technique viable for large data sets. This gives nearly identical output to the full method. These methods demonstrate substantial decreases in data rejection compared to the use of a priori parameters. These methods are viable for any application where maximum gradient contours must be accurately extracted from images of species or temperature, even at very low signal-to-noise ratios.

Detection of Copper Ion with Laser-Induced Fluorescence in A Capillary Electrophoresis Microchip

A capillary electrophoresis microchip coupled with a confocal laser-induced fluorescence (LIF) detector was successfully constructed for the analysis of trace amounts of heavy metals in environmental sources. A new fluorescence dye, RBPhOH, synthesized from rhodamine B, was utilized in a glass microchip to selectively determine copper with high sensitivity. A series of factors including running buffer concentration, detection voltage, and sample loading time were optimized for maximum LIF detector response and, hence, method sensitivity.

Capillary electrophoretic analysis of arsenic species with indirect laser induced fluorescence detection

The development of a method for determining arsenic species by capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence (LIF) is described in this paper. The buffer pH, the concentration of fluorescein, the nature and the concentration of the background electrolytes (BGEs) were defined. When 2.0 mM NaHCO3 (pH 9.28) with 10(-7) M fluorescein was used as the buffer, arsenite (As(lll), dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), and arsenate (As(V)) were all separated from one another. The limits of detection for the four arsenic species were p p in the range of 0.12-0.54 mg/L. This method was used in the analysis of spiked arsenic species in tap and mineral water to demonstrate its usefulness. The results showed that both the recovery and the reproducibility of the developed method were acceptable.