Heinz Detlef Kronfeldt

Detection of chemicals in seawater using surface-enhanced Raman scattering (SERS)

The development of surface-enhanced Raman scattering substrates suitable for in situ environmental analysis is presented. Sol-gel techniques are introduced to improve the mechanical stability of an existing active substrate type, to develop a new type of SERS active medium and finally to improve the sensitivity of substrates by providing a controllable coating. Two substrate types are discussed. The first consists of a glass slide covered with aluminium oxide particles which is then coated with gold.In the second type, a gold metal colloid is encapsulated within a porous thin film sol-gel structure. The sol-gel substrates were optically characterized and both substrates were tested in the laboratory. The mechanical stability of the new substrates is compared with that of conventionally prepared substrates and results are presented for pyridine, toluene and chlorobenzene in artificial sea-water.

Development of a SERS optode for the in-situ detection of chemicals in sea water

The purpose of this work is the development of a method for the in-situ detection of chemicals in sea water. Raman scattering was chosen as it is a non-intrusive method yielding finger-printing spectra of the analytes. Surface-enhanced Raman scattering (SERS) is used to provide the sensitivity necessary for trace analysis in the environment. SERS active coatings are produced by encapsulating a silver colloid in sol-gel derived xerogel films. The sol is coated onto silicon plates, microscope slides, quartz windows or optical fibers allowing a variety of measurement configurations. Results are presented for a range of aromatic hydrocarbons in sea water measured with methyl triethoxy silane (MTEOS) and ethyl triethoxy silane (ETEOS) based substrates. The suitability for operation in the marine environment is discussed in terms of selectivity, response time, limits of detection, and long term stability. Two compact marinised optodes are presented with a lensed head and a fiber head allowing for in-situ measurements in sea water with exchangeable SERS substrates or coated fibers. The optode is linked via optical fibers to an underwater core instrument containing light source and spectrograph. This is discussed in the context of a multi- disciplinary field-operable measuring device intended for operation depths up to 300 m.

Piezo-enhanced multireflection cells applied for in-situ measurements of trace-gas concentrations

We constructed a Herriott multipass cell with one piezo driven mirror to reduce Fabry-Perot- interference fringes in a pulsed infrared tunable diode laser spectrometer. The applicability of the cell is demonstrated by measuring weak lines in the (nu) 3-band of NO2 with a pulsed tunable diode laser spectrometer. The reduction of the fringes is discussed in dependence to the frequency of laser excitation, the frequency of the piezo-element, and the elongation of the piezo driven mirror. The multipass cell should become a part of a high sensitive TDL spectrometer for environmental research.

Characterization of endogenous fluorophores by picosecond laser fluorescence spectroscopy

By time resolved fluorescence spectroscopy in the psec range the fluorescence behavior of flavines as important endogenous fluorophores was investigated. The substances were examined under various conditions (e.g. pure solutions and cellular suspensions; different buffer systems and pH values). Particular attention was dedicated to the properties of these coenzymes under in situ conditions. The results were applied to test numerical methods for characterization of complex mixtures of fluorophores and for discrimination of different states of flavines.

NADH-Fluoreszenzspektroskopie in der medizinischen Diagnostik

Die NADH-Laserfluoreszenzspektroskopie bietet gunstige Moglichkeiten fur die Diagnostik des Energiestoffwechselzustandes von Zellen und Geweben, da das in diesen naturlicherweise vorkommende NADH, die reduzierte Form von NAD (Nicotinamid-Adenin-Dinukleotid), einerseits als wasserstoffubertragendes Coenzym selbst masgeblich an der Energiegewinnung auf zelluarer Ebene beteiligt ist und andererseits einen fur die Spektroskopie geeigneten naturlichen Fluoreszenzfarbstoff darstellt. Dieser kann durch Licht im nahen UV bei 337 nm angeregt werden und liefert eine Fluoreszenz im Blauen bei 470 nm.

Diode laser-diode laser-difference frequency spectrometer for the midinfrared spectral region

In the mid-IR spectral region between 3 micrometers and 25 micrometers lead salt diode lasers are the most common light sources for high-resolution spectroscopy. Other light sources as CO-, CO2-lasers or sideband-lasers are expensive, non- portable and poorly tunable. To overcome the cryogenic cooling of the lead salt diode lasers and the describe disadvantages of the other light sources the generation of tunable laser radiation by difference frequency generation in nonlinear crystals as an alternative method is well known. Because of the low efficiency of the nonlinear process dye, solid state or gas lasers are the preferred pump sources for difference frequency generation up to now. To use the flexibility of the nonlinear process with the advantages of the visible- or near-IR-diode lasers we developed a portable tunable cw difference frequency spectrometer applying the nonlinear crystal AgGaS2 and two diode lasers as pump sources. High resolution scans over CO, N2O and OCS absorption lines near 2107 nm-1 demonstrate the capabilities of the spectrometer. Moreover, we present high resolution line broadening measurements of the R(2) line of 13C16O in the fundamental band as well as experiments for trace gas detection of 12C16O in the fundamental band.

Application of a tunable spectrometer based on difference frequency generation in AgGaS 2 for line-broadening experiments on NO in the 5-μm region

The application of nonlinear crystals which enable the difference frequency generation (DFG) of two input lasers is a promising tool for light sources in the mid-infrared spectral region beside the well known lead salt diode laser and sideband CO- and CO2-lasers. We present a high resolution spectrometer based on the DFG in the nonlinear crystal AgGaS2 applying an AlGaAs index-guided single mode diode laser and a ring dye laser as pump sources. With an input power of only 5 mW (diode laser) and 150 mW (dye laser) by applying non-critical type I 90 degree phase matching, it was possible to achieve an infrared output power of 15 nW. Applying diode lasers in the wavelength region between 770 nm and 785 nm and a dye laser with DCM the wavenumber range from 1920 cm-1 to 2020 cm-1 could be covered. The typical phase-matching bandwidth was about 1 cm-1. Thus it was possible to measure several NO (Lambda) -doublets. For the doublet 2II3/2R(25.5) the line broadening for collisions with NO, N2, He, Ne, Ar, and Kr was investigated. The values for the self- and air broadening of this doublet, not studied previously, are smaller than those measured for lines with smaller quantum number. This corresponds to the known tendency that the broadening coefficient decreases with increasing quantum number.

Fiber optic approach to the fluorescence spectroscopy of cell cultures

A fiber-optical spectrometer for pharmacologically oriented energy metabolism studies using NADH fluorescence has been built up. Starting from theoretical considerations, the system parameters have been optimized. With this optimized geometry, the sensitivity limit for quantitative NADH detection has reached 20 (mu) M. The absolute lower detectable concentration value limited by SNR and other background effects lies below 5 - 10 (mu) M. With cultures of endothelial cells, measurements of the age dependence of NADH fluorescence intensity have been performed. They give a criterion for the metabolic activity of cells selected for further pharmacological investigations.

Studies of the metabolism of cell cultures by microspectrofluoroscopy

The monitoring of the state of cellular energy metabolism and respiratory activity is a necessary procedure in cell biology and pharmacology. One method is the observation of the redox state by NADH and FAD autofluorescence measurements. Using this technique, investigations on endothelial cell cultures were done to study their behavior under pharmacologic influences. One application was the investigation of cytotoxicity of cyanides, blocking the mitochondrial respiratory chain. Further we studied the activation of energy metabolism as a step of the cellular reaction on extracellular impacts. The measurements have been performed with a fluorescence microscope Zei(beta) Axioplan, extended by a PMT and a CCD camera. During examination, the cell cultures were kept under nearly physiological conditions using a specialized perfusion chamber. The measurements took place on cellular monolayers. Different excitation geometries have been studied to overcome the difficulties, which arose from the very weak absorption of the cell monolayer, resulting in a low quantum yield and SNR. In classical cytotoxicity studies, only the statistical long-time effects (e.g. IC50) of cell damages are recorded. By redox microspectrofluorometry it is possible to observe the process of damage in its progress, shown by the presented results. In the second, more complex model, we studied the reaction of cells on ligands like PIA (Phenylisopropyladenosin). In this case, the intracellular reaction is connected with an increased production of cAMP. Again, this requires an increased production of ATP, which leads to an activation of the cellular energy metabolism. The spectroscopic results are interpreted by a first model.

Registration of damages of endothelial cell cultures by beta-nicotinamide adenine dinucleotide (NADH) fluorescence

NADH is an indispensable mediator of energy metabolism in cells. By laser fluorescence spectroscopy with an experimental setup we studied the influence of different concentrations of NaCN solutions on the NADH fluorescence intensity of endothelial cell cultures of the calf aorta (BKz-7). The results obtained are discussed against the background of published cytotoxicity studies of cyanides with endothelial cell cultures.