Why is the propagation velocity of a photon in a transparent medium reduced?

A path integral formalism is used to describe the propagation of photons through a transparent medium. It is shown that the reduced phase velocity of light can be understood quantitatively by taking into account the contribution of all the possible classical paths the photon could have taken in order to reach a detector. These paths include all the multiple scattering processes by the atoms in the medium

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

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. \ud 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 s and resulting field strengths of 2.0 and 3.2 X 106 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.). \ud 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%