Karsten Koenig

Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NAD(P)H fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior-probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the time range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component as most pronounced for the intact strain DL1. Time-resolved fluorescence seems therefore to be an appropriate method to probe the function of the respiratory chain and-in the further step-to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin

Human skin shows a strong autofluorescence in the red spectral region with main peaks around 600, 620, and 640 nm caused by the porphyrin production of the gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of photoproducts with fluorescence bands around 670 nm and decay times of about 1 and 5 ns. The photoproduct formation is connected with an increased absorption in the red spectral region. The endogenous fluorescent porphyrins act as photosensitizers. Photodestruction of Propionibacteria acnes by visible light appears therefore to be a promising therapy. The photodynamic activity of the photoproducts is lower than that of protoporphyrin IX.

Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria

Carious regions of human teeth were investigated by means of remission-, fluorescence excitation- and fluorescence emission spectroscopy as well as time-resolved fluorescence measurements in the ns region. A detection of dental caries is possible with all of these spectroscopic methods. The peaks in measured remission- and fluorescence excitation spectra of carious regions correspond with the absorption bands of metal free porphyrins. The emission spectra and fluorescence decay kinetics indicate that protoporphyrin IX may be the main chromophore. HPLC measurement verify the existence of proto- (main component), copro- and probably Zn-protoporphyrin.

Time-gated microscopic imaging and spectroscopy in medical diagnosis and photobiology [also Erratum 33(11)3828(Nov1994)]

An experimental setup was developed for time-gated (nanosecond) fluorescence spectroscopy and imaging of microscopic samples. This makes it possible to depict individual components of complex fluorophores and to measure specific metabolites on the basis of their decay times. The field of applications includes the selective detection of intrinsic fluorophores and of photosensitizers in single cells, skin, and teeth, as well as of photosynthetic pigments in plants under various stress conditions.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after ALA administration using 633-nm light as therapeutic and fluorescence excitation radiation

Photodynamic therapy (PDT) and on-line fluorescence spectroscopy were carried out on human tumors after 5-aminolevulinic acid (ALA) administration using 633-nm light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the advantages of (1) enabling use of one laser for PDT and fluorescence diagnosis only, (2) enabling the possibility of on-line fluorescence measurements, and (3) exciting protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine excitation and fluorescence photon distribution in case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of excitation wavelength. The high penetration depth of 633-nm radiation results in a higher ratio of the 700-nm protoporphyrin fluorescence of the xenotransplanted tumor It to the skin Is compared with 407-nm excitation. No values greater than 1 for the ratio It/Is were found in the case of intravenous ALA injection even for red excitation. Therefore, a large amount of ALA will be metabolized in the skin and can cause photosensitivity of the patient when applied systematically. In contrast, protoporphyrin fluorescence limited to the pretreated skin area was detected in case of topically applied ALA to patients with mycosis funcoides and erythroplasy of Queyrat. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitivity fluorescence measurements.

Laser-induced autofluorescence of cells and tissue

The laser induced in-vivo autofluorescence in the visible spectral region of cells and tissues was studied using stationary and time-resolved fluorescence measurements as well as video- intensified- and confocal laser-scanning microscopy. Due to their excitation and emission spectra and their decay kinetics (ps- and ns-region) at least three intrinsic fluorophores were so far differentiated and attributed to the reduced pyridine coenzymes NADH/NADPH, free and bound flavin molecules and endogenous porphyrins. It was found, that defects in the mitochondrial respiratory chain as well as photodynamically or chemically induced cell destruction results in an increase of the fluorescence intensity in the blue/green spectral range.

Photodynamic tumor therapy and on-line fluorescence spectroscopy after aminolevulinic acid administration using 633-nm light as therapeutic and fluorescence excitation radiation

PDT and on-line fluorescence spectroscopy were carried out on human tumors after ALA- administration using 633 nm-light of a dye laser as therapeutic radiation and as fluorescence excitation radiation. This has the following advantages: (1) use of one laser for PDT and fluorescence diagnosis only, (2) the possibility of on-line fluorescence measurements, and (3) excitation of protoporphyrin molecules in deep tissue layers. Monte Carlo calculations were carried out to determine the excitation and fluorescence photon distribution in the case of red and violet excitation radiation. The results show the possibility of depth-resolved measurements on the fluorophore distribution by variation of the excitation wavelength. The influence of remitted excitation light and of the spontaneous radiation from the laser as well as the possible excitation of food-based degradation products of chlorophyll has to be considered in high-sensitive fluorescence measurements.

Optical determination of motility forces in human spermatozoa with laser tweezers

Laser tweezers may act as optical force transducers. We report on the determination of intrinsic motility forces of human spermatozoa by employing an 800 nm optical trap. The cellular forces were calculated from calibrated trapping forces. The determination of trapping forces based on a hydrodynamic model for ellipsoidal specimens, the measurement of the minimum laser power required to confine a single cell in the trap, and the calculation of viscus forces during the movement of optically trapped sperm heads through a laminar fluid. A mean motility force of 44 plus or minus 24 pN was calculated for spermatozoa of healthy donors.

Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap (laser tweezers)

We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into an inverted confocal laser scanning microscope. Fluorescence of cells in the single- beam gradient force optical trap was excited with a 488 nm microbeam (laser scanning microscopy) or with 365 nm radiation from a high- pressure mercury lamp. Modifications to NADH-attributed autofluorescence and Rhodamine- and Propidium Iodide-attributed xenofluorescence indicate a significant cell-damaging effect of 760 nm trapping beams. 760 nm effects produce a biological response comparable to UVA-induced oxidative stress and appear to be a consequence to two-photon absorption.

Studies on porphyrin photoproducts in solution, cells, and tumor tissue

Light excitation of photosensitizing porphyrins leads to cytotoxic reactions. In addition, photobleaching and photoproduct formation occur indicating photosensitizer destruction. Photoproducts from hematoporphyrin (HP) fluoresce in aqueous solution at 642 nm, whereas photoproducts from protoporphyrin (PP) in hydrophobic environment emit around 670 nm and exhibit pronounced absorption at 665 nm. Photoproduct formation depends on singlet oxygen. The photoproducts exhibit faster fluorescence decay kinetics compared with nonirradiated porphyrins, as shown by time-grated spectroscopy and fluorescence decay measurements. Photoproduct fluorescence was observed during light exposure of cells and of tumor-bearing, nude mice, following administration of Hematoporphyrin Derivative (HpD), tetramethyl-HP, and PP. Photoconversion was also detected with naturally-occurring porphyrins (PP-producing bacteria) and ALA-simulated biosynthesis of PP in tumor tissue and in skin lesions of patients (psoriasis, mycosis fungoides). The efficiency of PDT with porphyrin photoproducts was found to be low in spite of the strong electronic transitions in the red spectral region.