Optics: measuring and testing – Blood analysis
Patent
1987-03-05
1988-04-12
McGraw, Vincent P.
Optics: measuring and testing
Blood analysis
356 72, 356 73, G01N 3348, G01N 2149, G01N 2164
Patent
active
047370254
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a device which is a flow chamber to be employed in flow cytometers. This device facilitates that the biological cells or other microscopical particles, carried by a microscopical laminar flow of fluid, are brought one by one across the open surface of a plane glass with a velocity which may be varied from above 30 m/sec. to below 1 cm/sec., so that the fluorescence and light scattering of the cells/particles may be measured through optics situated on each side of the plane glass. A further development of the device facilitates that the electrical volume of the cells/particles may be measured simultaneously.
The function of the flow chamber in such instruments is to make particles or biological cells to flow one by one in a highly reproducible manner through the measuring area of the instrument.
The intention of the present invention is to facilitate considerably lower flow velocity and a corresponding increase in the detection sensitivity as compared to other types of flow chambers and furthermore to facilitate the measurement of the electrical volume of the particles/cells simultaneously with meaurement of the photometrical signals.
Flow cytometers are instruments for the measurement of the fluorescence and light scattering properties of microscopical particles. The measurement is carried out when the particles, carried by a laminar flow of fluid of microscopical dimensions, pass one by one through the focus of a beam of excitation light. The pulse of fluorescence emitted by each particle when it passes through this focus is determined by the chemical composition of the particle and is characteristic of this composition. The pulses of fluorescence are detected through appropriate optics by a photoelectric detector which transforms the pulses of fluorescence to equivalent electrical pulses. These pulses are transferred to a multichannel pulse height analyzer (MCA) which measures the size of the pulses and thereby produces a histogram showing the number of particles as a function of fluorescence intensity.
The light which is scattered by each particle when it passes through the focus of excitation light, and which is determined by the structure and the size of the particle, is detected in a similar manner by another detector and thus gives rise to a histogram which characterizes the particles with regard to structure and size.
The fluid flow in a flow cytometer may have a typical velocity of about 20 m/sec., and the excitation focus which constitutes the measuring area of the instrument may have a size of about 50 .mu.m. This means that each particle is measured in about 3 .mu.sec., which in turn means that it is possible to measure several thousand particles per second. The smallest amount of fluorescent material per particle which may be detected is about 1.multidot.10.sup.-16 g. Particle sizes below 1.multidot.10.sup.-2 .mu.m.sup.3 may be measured by light scattering. Both fluorescence and light scattering may be measured with a precision of about 1%. The most common use of flow cytometers has so far been measurement of the size of biological cells and their content of essential components such as DNA, enzymes and other proteins.
The flow chamber is a critical component in all flow cytometers because the stability of the particle flow with regard to position as well as velocity determines the precision of the measurement. In order to achieve high stability most flow chamber employs hydrodynamic focusing of the particles. Hydrodynamic focusing is achieved when the carrying fluid is forced throgh the microscopical orifice of a conical nozzle so that a laminar jet of fluid is produced. The particles are introduced in the carrying fluid through a thin cylindrical tube having its axis in common with that of the nozzle and its opening where the nozzle diameter is much larger than the diameter of the cylindrical tube. Thus, the particles will be centered in the microscopical jet which leaves the nozzle.
In one type of flow chambers the free jet produced by the nozzle is used as me
REFERENCES:
patent: 4225229 (1980-09-01), Gonde
patent: 4408877 (1983-10-01), Lindmo et al.
patent: 4426154 (1984-01-01), Steen
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