Wolfgang Hoehne

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.

Noninvasive NADH measurements for clinical applications

Although NAD/NADH and their spectroscopical characteristics have been well known for a relatively long time, the use of NADH-fluorescence measurements for clinical diagnostics is rather rare up to now Probing the application of a multifiber-NADH-fluorescence-measuring-apparatus in different fields of clinical research and clinical practice, the wide span of applicability is demonstrated. The main purpose of NADH-fluorescence diagnosis is to monitor the well-functioning of the respiratory chain, i.e. the energy metabolic situation of the tissue area probed. As a typical example, the observation of the metabolic state of tissue in transplantation surgery is considered on the one hand and the usefulness of the topological recognition of tumor areas on the other hand.

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.

NADH- and FAD-fluorescence measurements on cultures of endothelial cells

After a short introduction on NADH and FAD as coenzymes of the respiratory chain a new method and the apparatus for evaluating cell damage are presented for the case that cultures of endothelial cells are treated with different concentrations of H2O2. This is of interest for research on reperfusion injuries and inflammatory processes.

Correction of blood perfusion influences on NADH fluorescence spectroscopic measurements

In vivo measurement of NADH autofluorescence is strongly disturbed by changes in the Hb absorption of excitation and fluorescence light. An approach to this problem is based on the diffuse reflectance measurement of 805 nm (isobestic point of Hb/HbO2). According a Kubelka-Munk calculation an increase in Hb/HbO2 concentration leads to a decrease of diffuse reflectance. This gives correction terms for the measured autofluorescence data. An equipment to perform such measurements will be presented.