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H Schneckenburger

Publications and source records attributed to H Schneckenburger.

At least 19 recordsLinked to original sources
PubMed2003

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device.

A novel compact illumination device in variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell-substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four-layer model, as well as a Gaussian-shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell-substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time-resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).

2-Naphthylamine↗
PubMed2003

Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the membrane marker laurdan.

A novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the membrane marker laurdan either selectively within the plasma membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time-gated detection with a delay of 10-15 ns with respect to the exciting laser pulse was used. As a parameter of membrane dynamics the so-called generalized polarization GP = (I440 - I490)/(I440 + I490) was evaluated at temperatures between 24 and 41 degrees C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma membrane or in the whole cell. A decrease of the GP values by approximately 0.2 units between 28 and 41 degrees C indicated an increase in membrane fluidity or a decrease in membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma membrane as compared with intracellular membranes, probably due to a higher amount of cholesterol. Because properties of the plasma membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.

2-Naphthylamine↗
PubMed2001

Optical detection of mitochondrial NADH content in intact human myotubes.

Time-gated fluorescence spectroscopy in combination with non-radiative energy transfer was used on intact human skeletal myotubes for the determination of the mitochondrial NADH content which is considered to be a sensitive indicator of mitochondrial function. To mimic dysfunction of the mitochondrial energy metabolism, complexes I or III of the respiratory chain were inhibited by drugs. In the absence of the fluorescent mitochondrial marker rhodamine (R123), the NADH autofluorescence (i.e. a signal monitoring cytoplasmic plus mitochondrial NADH) remained unchanged upon inhibition of complex I by rotenone, and was increased by a factor of 2 upon inhibition of complex III by antimycin. In the presence of R123, the autofluorescence of NADH was reduced indicating non-radiative energy transfer from NADH to R123. The ratio of the R123 fluorescence signals obtained with the two excitation wavelengths of 355 nm and 488 nm was taken as a measure of mitochondrial NADH. Relative NADH changes were estimated in the presence of the above-mentioned inhibitors. Upon complex I inhibition, mitochondrial NADH was increased by a factor of 1.5. Upon inhibition of complex III, mitochondrial NADH was increased by a factor of 2. We conclude that time-gated spectroscopy combined with non-radiative energy transfer is an appropriate tool for probing mitochondrial enzyme complex deficiencies.

Antimycin A↗
PubMed2000

Statoliths in Phycomyces: characterization of octahedral protein crystals.

To identify the molecular mechanisms of gravitropism in the fungus Phycomyces blakesleeanus we determined several biochemical and physical parameters of paracrystalline protein bodies, so-called octahedral crystals. The crystals, which are present throughout the central vacuoles of the sporangiophore, function as statoliths (Schimek et al., 1999a,b). They possess an average volume of 9.96 microm(3) and a specific mass of 1.26 g cm(-3). SDS-PAGE of purified crystals shows three major proteins with relative molecular masses of 16, 46.5, and 55 kDa. These proteins are absent in gravitropism mutants which lack the crystals. Phototropism mutants (genotype mad) which are graviresponsive (class 1) and those which are defective in gravitropism (class 2) contain the crystals and the three associated proteins. Absorption spectra of isolated crystals and in situ absorption spectra of growing zones indicate the presence of chromophores, probably oxidized and reduced flavins. The flavin nature of the chromophores is also indicated by their fluorescence properties. It appears likely that the chromophores represent an essential part of the statoliths and thus the gravitropic transduction chain.

Crystallization↗
PubMed2000

Metabolic adaptations to environmental changes in Caenorhabditis elegans.

Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20 degrees C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20 degrees C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10 degrees C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

A quotient of independent measurements as outcome. A statistical method to compare groups in biological experiments.

The methodology in this paper was proposed to analyze biological data. The variable of interest, W, was a quotient of measurements; W: = X/(Y.Z). The peculiar problem was that X, Y, and Z could only be determined in three independent units of observation which were destroyed in the course of each measurement. A test statistic for the comparison of two treatments in W is proposed which is based on robust measures of location and dispersion in order to account for outliers in the data. Simulations showed that the test statistic proposed is approximately normally distributed, even for small sample sizes.

Algorithms↗
PubMed2000

Time-resolved in situ measurement of mitochondrial malfunction by energy transfer spectroscopy.

To establish optical in situ detection of mitochondrial malfunction, nonradiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. A screening system was developed in which these signals are detected simultaneously using a time-gated (nanosecond) technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the apparatus are described, and results obtained from cultivated endothelial cells are reported. The ratio of fluorescence intensities excited in the near ultraviolet and blue-green spectral ranges increased by a factor 1.5 or 1.35 after inhibition of the mitochondrial respiratory chain by rotenone at cytotoxic or noncytotoxic concentrations, respectively. Concomitantly the amount of mitochondrial NADH increased. Excellent linearity between the number of cells incubated with R123 and fluorescence intensity was found in suspension.

Animals↗
PubMed2000

Cell viability in optical tweezers: high power red laser diode versus Nd:YAG laser.

Viability of cultivated Chinese hamster ovary cells in optical tweezers was measured after exposure to various light doses of red high power laser diodes (lambda = 670-680 nm) and a Nd:yttrium-aluminum-garnet laser (lambda = 1064 nm). When using a radiant exposure of 2.4 GJ/cm2, a reduction of colony formation up to a factor 2 (670-680 nm) or 1.6 (1064 nm) as well as a delay of cell growth were detected in comparison with nonirradiated controls. In contrast, no cell damage was found at an exposure of 340 MJ/cm2 for both wavelengths, and virtually no lethal damage at 1 GJ/cm2 applied at 1064 nm. Cell viabilities were correlated with fluorescence excitation spectra and with literature data of wavelength dependent cloning efficiencies. Fluorescence excitation maxima of the coenzymes NAD(P)H and flavins were detected at 365 and 450 nm, respectively. This is half of the wavelengths of the maxima of cell inactivation, suggesting that two-photon absorption by these coenzymes may contribute to cellular damage. Two-photon excitation of NAD(P)H and flavins may also affect cell viability after exposure to 670-680 nm, whereas one-photon excitation of water molecules seems to limit cell viability at 1064 nm.

Absorption↗
PubMed2000

Plasma membrane associated location of sulfonated meso-tetraphenylporphyrins of different hydrophilicity probed by total internal reflection fluorescence spectroscopy.

Sulfonated meso-tetraphenylporphyrins of different hydrophilicity were microspectrofluorimetrically examined in endothelial cells using total internal reflection (TIR) illumination or epi-illumination. Since the penetration depth of the evanescent field during TIR illumination is limited to a few hundred nanometers, photosensitizers were almost selectively examined in close vicinity to the plasma membrane. Pronounced fluorescence signals during TIR illumination were observed for the hydrophilic compounds meso-tetraphenylporphyrin tetrasulfonate (TPPS4) and meso-tetraphenylporphyrin trisulfonate (TPPS3), whereas the more lipophilic compounds meso-tetraphenylporphyrin disulfonate (TPPS2a) and meso-tetraphenylporphyrin monosulfonate (TPPS1) could only be detected under epi-illumination. Irradiation of TPPS1 and TPPS2a in the Soret band led to an increase in fluorescence intensity and formation of a photoproduct with an emission maximum around 610 nm, which was limited to intracellular compartments. In contrast, fluorescence spectra of TPPS3 and TPPS4 obtained by TIR and epi-illumination remained almost unchanged after irradiation in the Soret band. Extralysosomal location of TPPS3 and TPPS4 in close proximity to the plasma membrane was deduced from experiments with the lysosomal markers acridine orange (AO) or lysotracker yellow (LY), which were not detectable under TIR illumination. In conclusion, these results provide for the first time direct evidence for a plasma membrane-associated fraction of the hydrophilic compounds TPPS3 and TPPS4 in living cells.

Cell Membrane↗
PubMed1999

Time-gated in vivo autofluorescence imaging of dental caries.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.

Dental Caries↗
PubMed1998

Selective examination of plasma membrane-associated photosensitizers using total internal reflection fluorescence spectroscopy: correlation between photobleaching and photodynamic efficacy of protoporphyrin IX.

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epi-illumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, whereas spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.

Animals↗
PubMed1998

Time-gated fluorescence microscopy in cellular and molecular biology.

An experimental set-up for time-gated fluorescence spectroscopy and microscopy is described, and some recent applications in cellular and molecular biology are summarized. Selective detection of intrinsic fluorophores, in particular nicotinamide adenine dinucleotide (NADH) and flavins was demonstrated in living cells. Non-radiative energy transfer from reduced NADH to the mitochondrial marker rhodamine 123 was evaluated for probing mitochondrial malfunction in living cells. An increase of "energy transfer efficacy" up to a factor 4 was detected after inhibition of enzyme complexes of the respiratory chain. Two different fluorescence lifetimes of calcium orange were evaluated, whose relative intensities depended on calcium concentration. Therefore, fluorescence measured within two different time gates appeared to be suitable for ratio fluorometry of calcium. Time-gated fluorescence spectra of the membrane marker laurdan showed more pronounced changes than steady state spectra when temperature was increased from 24 degrees C to 38 degrees C. This may improve measurements of intracellular temperature. Time-gated detection of small amounts of porphyrins and their discrimination from a large fluorescent background caused by chlorophyll in transgenic tobacco plants again proved the advantages of time-gated fluorescence spectroscopy.

2-Naphthylamine↗
PubMed1997

Photodynamic efficacy of naturally occurring porphyrins in endothelial cells in vitro and microvasculature in vivo.

Photodynamic therapy (PDT) has been described in terms of cellular and vascular effects. The precise mechanisms of cellular and vascular damage are still unknown. In this study, the photodynamic inactivation of endothelial cells in vitro and damage to the microvasculature in vivo by naturally occurring porphyrins (uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP)) were investigated. The chick chorioallantoic membrane model (CAM model) was used, which is convenient for the study of damage to the microcirculation induced by PDT. The hydrophilic porphyrins UP and CP exhibited low cytotoxicity towards endothelial cells. Only small amounts of UP and CP were taken up, resulting in weak inactivation after irradiation. In contrast, the more lipophilic PP showed a marked cytotoxicity. Considerable amounts of PP were accumulated in the cells, leading to pronounced inactivation after light exposure. For the three porphyrins, damage to the microvasculature was observed. The damage caused by the hydrophilic porphyrins UP and CP was strongly dependent on the drug and light dose. For vascular injury, the efficacy was graded as UP < CP < PP.

Animals↗
PubMed1997

Energy transfer spectroscopy for measuring mitochondrial metabolism in living cells.

Microscopic energy transfer spectroscopy was established using mixed solutions of reduced nicotinamide adenine dinucleotide (NADH) and the mitochondrial marker rhodamine 123 (R123). This method was applied to probe mitochondrial malfunction of cultivated endothelial cells from calf aorta incubated with various inhibitors of specific enzyme complexes of the respiratory chain. Autofluorescence of the coenzyme NADH as well as energy transfer efficacy from excited NADH molecules (energy donor) to R123 (energy acceptor) were measured by time-gated fluorescence spectroscopy. Because intermolecular distances in the nanometer range are required for radiationless energy transfer, this method is suitable to probe selectively mitochondrial NADH. Autofluorescence of endothelial cells usually exhibited a weak increase after specific inhibition of enzyme complexes of the respiratory chain. In contrast, pronounced and statistically significant changes of energy transfer efficacy were observed after inhibition of the same enzyme complexes. Detection of NADH and R123 in different nanosecond time gates following the exciting laser pulses enhances the selectivity and improves quantification of fluorescence measurements. Therefore, time-gated energy transfer spectroscopy is suggested to be an appropriate tool for probing mitochondrial malfunction.

Animals↗
PubMed1996

Influence of rhodamine 123 on the photosensitizing properties of porphyrins.

The photophysical and photochemical properties of porphyrins were profoundly changed upon addition of rhodamine 123. The Soret band of the porphyrins shifted to higher wavelengths, the fluorescence yield of the porphyrins decreased with unaltered decay rates, and their triplet state was quenched. These observations indicate a strong interaction between porphyrins and rhodamine 123 and formation of 1:1 nonfluorescent complexes, of which the binding constants were determined. Illumination of a porphyrin in the presence of rhodamine 123 resulted in the formation of a porphyrin radical cation, which could be detected with ESR spectroscopy. Quenching of the triplet state of the porphyrins by rhodamine 123 resulted in a decreased singlet oxygen yield and a decrease of the photooxidation of histidine, methionine, tyrosine, and tryptophan. However, the oxidation of thiol compounds was increased and the stoichiometry of the reaction between cysteine and oxygen changed from 2 to 3.8 mol cysteine/ mol oxygen. These results show that the presence of rhodamine 123 converted the for porphyrins prevalent energy transfer (type II) reaction to an electron transfer (type I) reaction.

Cations↗
PubMed1995

Intracellular fluorescence behaviour of meso-tetra(4-sulphonatophenyl)porphyrin during photodynamic treatment at various growth phases of cultured cells.

Meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) taken up by cells is mainly localized in lysosomes as previously shown by fluorescence microscopical and fluorescence spectroscopical investigations. In the present study the intracellular fluorescence behaviour and the intracellular amount of this dye at various growth periods of cells were examined. For cells irradiated in the growth phase a relocalization of TPPS4 from the lysosomes into the cytoplasm and finally into the nucleus was observed. In contrast, for cells irradiated in the stationary phase no redistribution could be detected and therefore no evidence for severe damage of the lysosomal membranes and subsequently for the release of lytical enzymes is given. In both cases lethal damage of the cells was achieved as examined using the trypan blue exclusion test. This indicates that damage of the lysosomes is less important in the photodynamic inactivation of cells sensitized by TPPS4.

Animals↗