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Biomedical subjects

J Greve

Publications and source records attributed to J Greve.

At least 19 recordsLinked to original sources
PubMed1998

Photon-counting device compatible with conventional flow cytometric data acquisition electronics.

We present an electronic scheme that enables us to use a photon-counting device (photomultiplier or avalanche photodetector) for measuring extremely weak signals in a flow cytometer. It can be used as a sole detector, or in combination with other (conventional) detectors using the data acquisition hardware of a conventional flow cytometer. The essential principle is that photon-counting pulses are converted to an analogue signal that is continuously proportional to the number of detected photons during the last integration time. The integration time should be approximately equal to the time an object is illuminated in the flow chamber. In this way, the photon burst due to real events is measured correctly and discriminated from the background pulses (fluorescence and Raman). The use of this scheme for the measurement of single DNA molecules is illustrated.

DNA↗
PubMed1998

New technique for high resolution DNA sizing in epi-illumination.

We present a high-resolution DNA-sizing technique based on the principles of flow cytometry, using a high numerical aperture objective and epi-illumination. The new technique, designed for small fluorescing samples/particles (sub-micron diameter) suspended in a weakly fluorescent medium, makes use of an additional focus for high-precision particle localisation. This way, only those particles are considered that flow exactly through a well-defined volume. Results are presented for fluorescent beads, as well as for YOYO-stained plasmids containing 5,500 basepairs. The latter were measured with 6.2% resolution, setting a new limit to flow-based sizing of DNA.

DNA↗
PubMed1998

The waveguide Mach-Zender interferometer as atrazine sensor.

Immunoanalytical techniques used in combination with a highly sensitive Mach-Zender waveguide sensor give a device that is capable of fast on-line monitoring of immunoreactions. The on-line monitoring is especially attractive when the sensor can be used as an environmental probe. It is demonstrated that low concentrations of atrazine can be measured with a sensitivity around the EC limit of 100 ng/L. These measurements are performed with an inhibition technique. In combination with "a dynamic slope method" one complete measurement is done within 10 min. Sensor measurements show the same sensitivity and errors as parallel ELISAs.

Atrazine↗
PubMed1998

Chromatin structure in bands and interbands of polytene chromosomes imaged by atomic force microscopy.

Polytene chromosomes from Drosophila melanogaster, observed from squash preparations, and chromosomes from Chironomus thummi thummi, investigated under physiological conditions, are imaged using an Atomic Force Microscope. Various chromatin fiber structures can be observed with high detail in fixed chromosomes and correspond to structures which are also observed in chromosomes of diploid cells. Unfixed chromosomes can be imaged in buffer and show less fiber-like details because of the inherent soft nature of the chromatin material.

Animals↗
PubMed1998

Confocal Raman microspectroscopy of the activation of single neutrophilic granulocytes.

Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both resting cells as well as activated cells were measured. The activation of cells was performed with phorbol-12-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a high spatial resolution inside a single, living cell with detailed material information. Using this approach it can be concluded that activation of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b558. This protein is a part of the NADPH-oxidase complex that neutrophilic granulocytes employ to generate O-2, superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react with O-2. Activation of the cells with bacteria gives rise to corresponding changes in the Raman spectra. From this single cell study it can be concluded that the enzymes cytochrome b558 and myeloperoxidase are present inside the cytoplasm of the living cell, while participating in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granulocytes from a patient with chronic granulomatous disease, that contain no cytochrome b558, led to typical changes in the redox state of myeloperoxidase. This indicates that in the bacterium/neutrophilic granulocyte system oxygen metabolites are generated that are capable of reacting with MPO.

Granulomatous Disease, Chronic↗
PubMed1998

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Aluminum Silicates↗
PubMed1998

Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope.

Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.

Binding Sites↗
PubMed1998

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.

Cytochrome b Group↗
PubMed1998

Cholesterol, phospholipid, and protein changes in focal opacities in the human eye lens.

PURPOSE: Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). METHODS: Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. RESULTS: Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobicity of tyrosine residues showed a significant decrease, and a change in the tryptophan indole ring angle was found. The changes were abrupt and sharply delineated focal opacities. TEM confirmed this sharp boundary and showed that the opacities were densely packed with vesicles of varying size and electron density embedded in a homogenous matrix. CONCLUSIONS: The Raman and TEM analyses of opacities showed that early cataractogenic events led to disruption of fiber membranes, formation of vesicles from the membrane constituents, and protein changes. The aberrant morphology of the membranes enveloping the focal opacities may have segregated the affected fibers from the surrounding normal tissue, thus explaining the stationary or slowly progressing character of these opacities.

Adult↗
PubMed1997

Experimental and model investigations of bleaching and saturation of fluorescence in flow cytometry.

We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein-isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate kb1 = 6 x 10(3) s(-1), triplet state population rate k12 = 2 x 10(5) s(-1), and depopulation rate k20 = 5 x 10(4) s(-1). The fluorescence kinetics of FITC- and PE-labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water.

Flow Cytometry↗
PubMed1997

Intracellular carotenoid levels measured by Raman microspectroscopy: comparison of lymphocytes from lung cancer patients and healthy individuals.

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells.

Adenocarcinoma↗
PubMed1997

On refraction in Monte-Carlo simulations of light transport through biological tissues.

To obtain reliable results from Monte-Carlo simulations of light scattering experiments, a statistically accurate procedure for positioning the photons after refraction between two different scattering media is necessary. Two statistically equivalent algorithms for calculating the position of the photons immediately after crossing an interface are described and justified.

Algorithms↗
PubMed1997

[Assessment methods for vocational integration of disabled persons--from ERTOMIS methods to the IMBA information system].

The background for the development of actual assessments for evaluation of vocational integration of disabled people (i.e., EAM, IMBA) in Germany is described. Resulting perspectives for future procedural approaches are presented. So far, the EAM, Ertomis Assessment Method, approach has gained limited acceptance only. In light of WHO and European impulses, as well as the economic constraints at hand, it remains to be seen whether the IMBA information system will turn out a practical and problem-focussed tool. A critical review is done to provide constructive suggestions for further applications of IMBA.

Disability Evaluation↗
PubMed1996

The critical sensor: a new type of evanescent wave immunosensor.

A new planar waveguide immunosensor has been developed in which adsorption at a surface, changing the refractive index contrast, is measured. In this "critical" sensor the change in the effective refractive index contrast is transducted to a shift of the critical reflection angle. The sensor's theoretically. In addition, an experimental sensitivity evaluation on the basis of several immunosensing experiments is presented. The obtained lower detection limit is 2 x 10(-2) nm in adlayer growth, equivalent to 12 pg/mm2 of analyte coverage. This sensitivity is comparable to the performance of the surface plasmon resonance sensors or the grating coupler sensors. However, the "critical" sensor has some advantages. These are mainly the ease of fabrication and adjustment prior to a measurement, and the fact that for an experiment no metal layer has to be used.

Antigen-Antibody Reactions↗
PubMed1996

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy.

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Biotin↗
PubMed1996

Aging affects the conformation of cholesterol in the human eye lens.

The distribution of 3-beta-OH-cholesterol along the optical axis of 20 human eye lenses of different ages was measured using confocal Raman microspectroscopy in combination with the 3-beta-hydroxysteroid-specific probe filipin. This non-destructive technique allow a spatially very detailed study of the distribution of 3-beta-OH-cholesterol in individual lenses. It revealed age-related changes of the 3-beta-OH-cholesterol distribution along the optical axis. The 3-beta-OH-cholesterol was found to be distributed asymmetrically along the optical axis and this distribution was found to change with age. The relative 3-beta-OH-cholesterol content in the anterior part of the lens decreases significantly with increasing age compared with its equal posterior counterpart. Additionally all the lenses have a very low 3-beta-OH-cholesterol content in the central part of their nuclear region, the area where the primary lens fibres are located.

Adolescent↗
PubMed1996

Toothpick perforation of the intestine diagnosed by a small bowel series.

We report the case of a 50-yr-old man who unknowingly swallowed a double-pointed toothpick that ultimately perforated the distal small bowel over a period of several weeks. Appendicitis and ileitis were sequentially considered as diagnosis. A foreign body was diagnosed on a second small bowel series and removed. It has been noted that, because toothpicks are not radiopaque, radiographic studies are not useful in the diagnostic workup. In the case reported, the toothpick was seen on the contrast radiograph of the small bowel and the correct diagnosis ultimately made.

Appendicitis↗