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 micropheres (diameter 3.8 µm) and a suspension of the unicellular algae chlorella vulgaris (diameter about 3 µm). It appears that the alignment of cells and spheres is very good. \ud When using microspheres, more than 95% of the particles in the grooves are located within ±2 µm 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. \ud 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. It is expected that considerable improvements may be obtained by using a more sophisticated optical detection system such as the tracking system in use in optical disk players

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. \ud 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. \ud 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 argonion 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

Optical-trapping micromanipulation using 780-nm diode lasers

We have designed and implemented an optical-trapping configuration that uses near-infrared laser diodes. The highly divergent output beam of the diode laser was collimated by using only one aspheric compact disc lens. The resulting output beams are astigmatic and elliptic and have a flat, non-Gaussian intensity profile. Calculations and measurements were performed to investigate the influence of this profile on the trapping forces. The results show that use of a laser diode, collimated with a compact disc lens, provides a near-infrared light source that can be used for optical trapping. The light source is compact and relatively cheap and can be easily incorporated into an existing microscope

Four-Parameter white blood cell differential counting based on light scattering measurements

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinephilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. \ud A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0° and 2.6° and angles between 3.0° and 11.0°. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. \ud Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes

Discrimination of human cytotoxic lymphocytes from regulatory and B-lymphocytes by orthogonal light scattering

Light scattering properties of human lymphocyte subpopulations selected by immunofluorescence were studied with a flow cytometer. Regulatory and B-lymphocytes showed a low orthogonal light scatter signal, whereas cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15 revealed a large orthogonal light scatter signal. Two populations in light scatter histograms could be observed with monoclonal antibodies directed against determinants present on both regulatory and cytotoxic lymphocytes. By analysis of the lymphocytes of 16 individuals we found a linear relation between the number of cells with a large orthogonal light scattering and the number of cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15. These observations demonstrate physical differences between cytotoxic lymphocytes and regulatory and B lymphocytes. Moreover, the results suggest a method to estimate the amount of cytotoxic lymphocytes without using monoclonal antibodies