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B G de Grooth

Publications and source records attributed to B G de Grooth.

47 records · Page 3Linked to original sources
PubMed1987

Light-scattering polarization measurements as a new parameter in flow cytometry.

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.

Blood Cells↗
PubMed1986

Physical discrimination between human T-lymphocyte subpopulations by means of light scattering, revealing two populations of T8-positive cells.

Light-scattering properties of human T-lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8-positive cells can be distinguished. The first population (T8a) has the same orthogonal light-scattering properties as T4-positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7-positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light-scattering properties of lymphocyte subpopulations in two patients with B-cell chronic lymphatic leukemia suggest that this observation is of clinical interest.

Antibodies, Monoclonal↗
PubMed1985

The cytodisk: a cytometer based upon a new principle of cell alignment.

A new method is described for one-dimensional alignment of small particles such as biological cells. A drop of the particle suspension is spread out on a flat disk or plate equipped with V-shaped grooves such as are present on a gramophone disk. After drying, the particles are located on the bottom of the grooves and are thus aligned in a one-dimensional array. The new alignment procedure is demonstrated with a suspension of fluorescent polystyrene microspheres (diameter 3.8 microns) and a suspension of the unicellular algae chlorella vulgaris (diameter about 3 microns). It appears that the alignment of cells and spheres is very good. When using microspheres, more than 95% of the particles in the grooves are located within +/- 2 microns of the centre line of the groove. Based upon this cell-alignment principle, a new cytometer, named the cytodisk, is proposed. The proposed system has a number of advantages over the flow cytometer, among which is the unique ability of relocating a previously measured cell for further measurement or visual examination. A prototype of a cytodisk, developed for initial test measurements, was built in our laboratory. The apparatus, constructed from a record player and ordinary long-playing records, uses a simple mechanical tracking system and a single optical fiber for fluorescence excitation and detection. With this apparatus it is demonstrated that a cytodisk can indeed perform quite well: A histogram of fluorescing microspheres could be measured with a coefficient of variation of 4.1%. The performance of this prototype is limited by the quality of the mechanical tracking system and the optical system used.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorella↗
PubMed1985

Flow cytometric determination of circulating immune complexes with the indirect granulocyte phagocytosis test.

A method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG-bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC-conjugated anti-human IgG was developed where the granulocytes remain in suspension during fixation and labeling. The fluorescence per cell, measured with a flow cytometer, is a measure of the total amount of the phagocytosed IgG. The results indicate that a rapid and quantitative method for the detection and measurement of phagocytosed CIC is possible using the flow cytometer.

Antigen-Antibody Complex↗
PubMed1981

External electric field effects on prompt and delayed fluorescence in chloroplasts.

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233-240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulate luminescence supported the interpretation given above. Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.

Chloroplasts↗
PubMed1980

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii.

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Qy) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Qy transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Qx transitions (590-620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.

Bacterial Proteins↗
PubMed1980

Electrochromic absorbance changes in spinach chloroplasts induced by an external electrical field.

Absorbance changes induced by electrical field pulses were studied in osmotically swollen spinach chloroplasts. The results and their interpretation on the basis of the geometry and electrical properties of the material may be summarized as follows: 1. The spherical vesicles, 'blebs', formed upon dilution of a chloroplast suspension consist of only a single membrane, while part of the thylakoid system remains concentrated in a few patches on its surface. 2. When an electrical field pulse is applied, an up to 3000-fold enhanced field is built up in the membrane, with a time constant of about 20 mus. From this the specific capacitance of the bleb wall was found to be 2 microF . CM-2. 3. The electrical field in the membrane causes several absorbance changes of the photosynthetic pigments with different dependencies on the direction of polarization of the measuring light. Some of these are due to field-induced changes in orientation, in particular of chlorophyll alpha, and have a relaxation time of less than 100 mus. Most of the absorbance changes directly reflect the kinetics of the membrane potential and can be ascribed to electrochromic shifts of photosynthetic pigments, mainly of carotenoids. 4. The carotenoid absorbance changes depend quadratically on the membrane potential; an apparent saturation at high applied field strengths is ascribed to dielectric breakdown at a membrane potential of about 1 V. 5. All carotenoids in the membrane contribute to the absorbance changes induced by an externally applied field, whereas the well-known light-induced electrochromic absorbance change at 518 nm is mainly caused by a minor fraction of permanently polarized and spectrally red-shifted carotenoids. A computer simulation showed that this interpretation quantitatively explains the results and requires no unreasonable values of the various parameters involved.

Carotenoids↗
PubMed1978

The mechanism of reduction of the ubiquinone pool in photosynthetic bacteria at different redox potentials.

(1) A flash number dependency of flash-induced absorbance changes was observed with whole cells of Rhodospirillum rubrum and chromatophores of R. rubrum and Rhodopseudomonas sphaeroides wild type and the G1C mutant. The oscillatory behavior was dependent on the redox potential; it was observed under oxidizing conditions only. Absorbance difference spectra measured after each flash in the 275--500 nm wavelength region showed that a molecule of ubiquinone, R, is reduced to the semiquinone (R-) after odd-numbered flashes and reoxidized after even-numbered flashes. The amount of R reduced was approximately one molecule per reaction center. (2) The flash number dependency of the electrochromic shift of the carotenoid spectrum was studied with chromatophores of Rps. sphaeroides wild type and the G1C mutant. At higher values of the ambient redox potential a relatively slow phase with a rise time of 30 ms was observed after even-numbered flashes, in addition to the fast phase (completed within 0.2 ms) occurring after each flash. Evidence was obtained that the slow phase represents the formation of an additional membrane potential during a dark reaction that occurs after flashes with an even number. This reaction is inhibited by antimycin A, whereas the oscillations of the R/R- absorbance changes remain unaffected. At low potentials (E = 100 mV) no oscillations of the carotenoid shift were observed: a fast phase was followed by a slow phase (antimycin-sensitive) with a half-time of 3 ms after each flash. (3) The results are discussed in terms of a model for the cyclic electron flow as described by Prince and Dutton (Prince, R.C. and Dutton, P.L. (1976) Bacterial Photosynthesis Conference, Brussels, Belgium, September 6--9, Abstr. TB4) employing the so-called Q-cycle.

Bacterial Chromatophores↗
PubMed1977

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature.

Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.

Bacterial Chromatophores↗
PubMedDate not supplied

Analysis of immunolabeled cells by atomic force microscopy, optical microscopy, and flow cytometry.

In this study we investigated the applicability of the (silver-enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied on these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a direct correlation was made between the force and the fluorescent images. Additionally, we performed flow cytometric analysis. From the results we conclude that immunogold labeling using small labels (1 nm) in combination with silver enhancement (30 min) proves to be a reliable method for high-resolution cell surface antigen detection in atomic force microscopy.

Animals↗