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Biomedical subjects

J Greve

Publications and source records attributed to J Greve.

At least 91 records · Page 5Linked to original sources
PubMed1992

Polarization sensitive coherent anti-Stokes Raman scattering spectroscopy of the amide I band of proteins in solutions.

Polarization sensitive coherent anti-Stokes Raman scattering (PCARS) spectroscopy is a fruitful technique to study Raman vibrations of diluted molecules under off-electron resonant conditions. We apply PCARS as a direct spectroscopic method to investigate the broad amide I band of proteins in heavy water. In spontaneous Raman spectroscopy, this band is not well resolved. We fit a number of spectra taken of each protein under different polarization conditions, with a single set of parameters. It then appears that some substructure is observed in the amide I band. From this substructure, we determine the percentage of alpha-helix, beta-sheet, and random coil for the proteins lysozyme, albumin, ribonuclease A, and alpha-chymotrypsin.

Amides↗
PubMed1992

A monitoring device for pressurised-air-driven diaphragm-based artificial heart assist devices.

A non-invasive device has been developed to monitor the diaphragm position and the blood flow in artificial heart assist devices equipped with a pressurised-air-driven diaphragm. Light scattering from the diaphragm is used as a mechanism for measuring. Information about the position of several points of the diaphragm can be obtained. The completely empty or filled situation can be detected and used for control purposes. Flow data can be extracted and bending characteristics of the diaphragm during operation can be studied.

Heart-Assist Devices↗
PubMed1992

Digital blood flow in cooled and contralateral finger in patients with Raynaud's phenomenon. Comparative measurements between photoelectrical plethysmography and laser Doppler flowmetry.

The effects of cooling of a hand on lateral and contralateral digital skin blood flow were investigated in 18 patients with primary or secondary Raynaud's phenomenon. The aim of the study was to compare photoelectrical plethsmography (PhEP) and laser Doppler flowmetry (LDF). PhEP and LDF were used simultaneously for skin blood flow measurements of the third finger of both hands. One hand was cooled in water from 33 degrees to 3 degrees C in steps of 3 degrees C, each step lasting four minutes. It was followed by a recovery period of ten minutes in room air of 24 degrees C. During stepwise cooling from 33 degrees to 9 degrees C the relative PhEP and LDF values, measured on the cooled hand, decreased to 6.2% +/- 3.2% and 10% +/- 12% respectively. The correlation coefficients between LDF and PhEP varied between 0.79 and 0.99. In the contralateral hand the relative PhEP and LDF values decreased to 38% +/- 30.% and 64% +/- 7.9% respectively. The correlation coefficients between LDF and PhEP values were lower on the contralateral hand and ranged from 0.26 to 0.95. By calculating the LDF/PhEP ratios during cooling and recovery, more specific changes in red blood cell velocities during cooling were studied. Increasing LDF/PhEP ratios suggest increasing red blood cell velocities during cooling at 9 degrees C and a difference in the measuring methods. For testing the severity of Raynaud's phenomenon and the effect of treatment the results of both methods show consistent and quite comparable results when measured on the cooled hand. The effect of indirect cooling on the contralateral hand is, however, less consistent.

Adult↗
PubMed1992

Reflectance pulse oximetry in fetal lambs.

Transmission pulse oximetry is used for monitoring in many clinical settings. However, for fetal monitoring during labor and in situations with poor peripheral perfusion, transmission pulse oximetry cannot be used. Therefore, we developed a reflectance pulse oximeter, which uses the relative intensity changes of the reflected red and infrared light (red/infrared ratio) to measure the arterial oxygen saturation. The performance of the reflectance pulse oximeter was studied in acute experiments in fetal lambs. By stepwise reduction of the inspired oxygen concentration of the ewe, measurements were done at the fetal scalp at various arterial oxygen saturation levels (17-82%). Reflectance pulse oximeter readings were averaged over periods of 15 s and compared with simultaneously taken fetal arterial blood samples. A calibration curve for the relationship between red/infrared ratio and arterial oxygen saturation was obtained from 53 measurements in four fetal lambs, by linear regression analysis [red/infrared = 4.088-(0.038.SaO2), r = 0.96]. In these experiments, the pulse oximeter showed a precision of 4.7% oxygen saturation around the calibration curve, with a 95% confidence interval of +/- 9.4%.

Animals↗
PubMed1991

Simple delay monitor for droplet sorters.

We have constructed a simple device by which the optimal delay time between optical measurement of a cell and the application of the droplet charging pulse can be determined directly in a flow sorter. The device consists of a stainless steel chamber in which the sorted droplets are collected. In the collection chamber the collected droplets run through a capillary where a continuous fluorescence measurement is made. With a sample of fluorescent particles, the delay time is optimal when the measured fluorescence is maximal. The measuring volume is always filled with the last droplets sorted (about 3,000). With this device, the setting of the delay time can be done in a few seconds without the need for microscopical verification. The fluorescence in the collection chamber is excited and detected via optical fibers using about 10% of the light of the existing laser from the flow cytometer and an extra photomultiplier.

Cell Separation↗
PubMed1991

Experimental and theoretical investigations on the validity of the geometrical optics model for calculating the stability of optical traps.

We have developed a computer program based on the geometrical optics approach proposed by Roosen to calculate the forces on dielectric spheres in focused laser beams. We have explicitly taken into account the polarization of the laser light and thd divergence of the laser beam. The model can be used to evaluate the stability of optical traps in a variety of different optical configurations. Our calculations explain the experimental observation by Ashkin that a stable single-beam optical trap, without the help of the gravitation force, can be obtained with a strongly divergent laser beam. Our calculations also predict a different trap stability in the directions orthogonal and parallel to the polarization direction of the incident light. Different experimental methods were used to test the predictions of the model for the gravity trap. A new method for measuring the radiation force along the beam axis in both the stable and instable regions is presented. Measurements of the radiation force on polystyrene spheres with diameters of 7.5 and 32 microns in a TEM00-mode laser beam showed a good qualitative correlation with the predictions and a slight quantitative difference. The validity of the geometrical approximations involved in the model will be discussed for spheres of different sizes and refractive indices.

Lasers↗
PubMed1991

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5 nm but not with 660 nm laser light.

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.

Animals↗
PubMed1991

Age-related changes in local water and protein content of human eye lenses measured by Raman microspectroscopy.

The Raman microspectroscopic method was used to determine the local water and protein content in human lenses. In 18 lenses of varying age position-defined water/protein content measurements were carried out along the visual and the equatorial axis. A main characteristic of the human lens is its constant and relatively low protein content. In addition this constant nuclear value is reached within a short distance from the capsular surface. For statistical analysis of age-related changes the data points in individual lenses were piecewise linearized. (1) The mean nuclear water content was calculated from the data points in the inner 80% of the visual axis. (2) The steep drop in water content was linearized using a least-squares linear regression approach. The distance between lenticular surface and the intersection of the regression line with the line representing the nuclear mean was denominated as surface layer width. It proved that: (i) the mean nuclear water content significantly increased with age, (ii) the width of the surface layer was age independent in the anterior and posterior poles of the visual axis, and (iii) in the equatorial axis the surface layer width significantly decreased with age. Seven human lenses with small opaque spots were also investigated. The opaque spots proved to have a normal-for-site water content and some of them were flanked at their capsular side by a zone with a high-for-site water content.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Raman microspectroscopic approach to the study of human granulocytes.

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Basophils↗
PubMed1990

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells.

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.

Cell Line↗
PubMed1990

Studying single living cells and chromosomes by confocal Raman microspectroscopy.

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

Animals↗
PubMed1990

A new principle of cell sorting by using selective electroporation in a modified flow cytometer.

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 microseconds and resulting field strengths of 2.0 and 3.2 x 10(6) V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%.

Cell Membrane Permeability↗
PubMed1990

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia. A flow cytometric study of peripheral blood, lymph nodes and bone marrow.

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD 57+ and cytotoxic CD 8+) wa studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7-27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD 57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.

Adult↗
PubMed1990

Skin blood flow changes, measured by laser Doppler flowmetry, in the first week after birth.

Changes in forehead skin blood flow were determined in 17 healthy, term newborns, using a fiberless diode laser Doppler flow meter (Diodopp). Measurements were carried out three times on each infant, at postnatal ages of 16.8 +/- 7.4 h, 58.9 +/- 6.2 h and 121.5 +/- 14.2 h (mean +/- S.D.), respectively. Skin blood flow, respiration, heart rate and skin temperature were recorded simultaneously, while the newborns were asleep. During the recordings, the behavioural state of the newborns was observed and environmental temperature and humidity were kept constant. Postocclusive hyperaemia of the skin blood flow was obtained by pressing the laser Doppler probe against the skull for 30 or 60 s. The following parameters changed significantly between the first and third measurements (t-test for paired samples): the basal skin blood flow during active and quiet sleep decreased, the average decrease being 29.4% (P = 0.002) and 25.9% (P = 0.01), respectively; skin blood flow during postocclusive hyperaemia also changed: the time taken to reach maximum hyperaemia increased from 17.3 to 22.7 s (P = 0.01), while the halftime recovery increased from 46.1 to 57.1 s (P = 0.02). The changes in skin blood flow between the first and second measurements and between the second and third measurements did not reach the level of statistical significance.

Blood Flow Velocity↗
PubMed1990

A new approach to immunoFET operation.

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.

Antigen-Antibody Reactions↗
PubMed1989

Local variation in absolute water content of human and rabbit eye lenses measured by Raman microspectroscopy.

Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm-1 divided by the Raman intensity at 2935 cm-1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens.

Animals↗
PubMed1989

Raman microspectroscopy of fixed rabbit and human lenses and lens slices: new potentialities.

Raman spectroscopy is a non-invasive, non-destructive technique for the study of the macromolecular composition of tissues. Raman spectra were obtained from intact fresh and paraformaldehyde fixed rabbit lenses and from thin slices prepared from these lenses. In addition the Raman spectrum of an intact 82-yr-old human lens was compared with a slice of the same lens. It appeared that fixation with paraformaldehyde had only a minor qualitative effect on the Raman spectra and that Raman spectra of intact lenses and lens slices were comparable. It was also shown that in the slice of the old human lens the fluorescence, due to chromophores, could be reduced so that a reliable Raman spectrum could be obtained. The use of slices improves the accuracy of the position at which Raman spectra are recorded and fixation extends the time available for Raman analysis which is particularly important for the study of human lenses. Moreover, slicing enables Raman analysis of old human lenses, which up to now was thought to be impossible due to the overwhelming fluorescence of the chromophores present in these lenses.

Aged↗