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C y t o m e t r y

Light Scattering

Light Scattering

Instrumentally Scanning Flow Cytometry is based on the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered light from individual moving particles (Flying Light Scattering Indicatrix, FLSI). The optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10° to 120° with integration at azimuthal angles from 0° to 360° and with angular resolution of better than 0.5°. The performance of the SFC has been demonstrated using certified polystyrene particles as reference material. The developed SFC, which, by recording the entire light scattering pattern of individual biological particles, would provide more useful information about the particle structure than the ordinary wide angle, forward and side scattering concepts. At present,the FLSI method has been developed in order to provide an absolute real-time determination of size and refractive index of individual spherical particles.

The Flying Light-Scattering Indicatrix of an individual lymphocyte. The ascertained refractive indices of polystyrene particles and lymphocytes correspond to published data. The indicatrix of the lymphocyte presented here is more realistic in comparison to the one introduced by Doornbos et al. (Applied Optics 35:729-734 (1996)).

Fluorescence

The fluorescent labels with long decay time (up to 1 ms) and time-resolved fluorescence detection have been found useful in reduction of the background fluorescence in bioaffinity assays. However, the ordinary flow cytometers are not applicable to time-resolved fluorescence. The measurement of long decay time fluorescence with a flow cytometer requires 1) excitation of the fluorochrome by pulsed light source, 2) measurement of the fluorescence intensity emitted by the particle as function of time. Pulsed nitrogen laser is suitable for excitation of labels with long decay time but a triggering system is required to start the laser. The design of the hydrofocusing optical cuvette of the first generation of the SFC, allows triggering of pulsed laser and recording of the decaying fluorescence emission from a moving particle, i.e. the time-resolved measurement of the fluorescence.

The photon collection efficiency of the optical cuvette of the Scanning Flow Cytometer as a function of particle location within the testing zone.

Maltsev et al. demonstrated an applicability of pulsed nitrogen laser in flow cytometry. The operation of a Pulsed Laser Flow Cytometer equipped by the pulsed nitrogen laser was tested from fluorometric analysis of human lymphocytes stained with FITC-labeled monoclonal antibodies. Using the SFC we studied the photon collection efficiency as a function of the distance from the spherical reflector bottom. For this purpose we used latex particles (size of 1.8 um) which were stained by pyronin and suspended them in a water in a concentration of 3*10^5 particles per ml. The collecting spatial angle depends on the particle location within the testing zone of the optical cuvette. The lens that focuses the fluorescence and the mask with the pin-hole were removed in these measurements. The fluorescence emitted from various particle locations within the optical cuvette was measured varying the delay time between the trigger laser signal and N2-laser start pulse. The maximum efficiency point for photon collection was found to be at a distance of 1.9 mm from the bottom of the spherical reflector. This fact is in agreement with calculation of the collection efficiency of the SFC optical cuvette (see Fig). The diaphragm diameter and location correspond to the diameter of the PMT photo-cathode and the PMT location, respectively. On basis of this result, one can optimize of SFC system properly, including the flow speed and the location of maximum efficiency point in relating to the decay curve for the purpose of measuring of long decay time fluorescent labels.

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