Optics: measuring and testing – By particle light scattering – With photocell detection
Patent
1987-03-06
1990-04-10
Rosenberger, Richard A.
Optics: measuring and testing
By particle light scattering
With photocell detection
356338, G01N 2100
Patent
active
049155016
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a device for simultaneous measurement of two angle components of the light scattering of biological cells or particles in flow cytometers comprising a microscope condenser with high numerical aperture containing a central field stop means which produces a conical dark field in the illumination field of said condenser, and a microscope objective with its light opening within said dark field and its object plane in common with the object plane of the condenser.
More precisely, the device facilitates measuring of the light scattering of biological cells and other microscopical particles in two different regions of light scattering angles as the cells or particles pass one by one through the measuring area of the flow cytophotometer. Because the light scattering within the respective angle regions depends on different characteristics of the cell/particle, viz. on one side the size (volume) and on the other side submicroscopical structure, such a device may be used to characterize and identify cells/particles which are different with regard to these characteristics.
Flow cytometry is a method which has numerous applications within various fields of cell biology, medicine as well as measurements of nonbiological particles of almost any material. In a flow cytometer photometrical signals from individual particles of microscopical size are measured as they, carried by a laminar flow of fluid, pass one by one through a focus of excitation light of high intensity. The particles are centered in the liquid flow by so called hydrodynamic focusing. The photometrical signals that can be measured are 1) the fluorescence of the particles/cells, 2) the light scattering of the the particles/cells, and 3) the light absorption of the the particles/cells. In particular the former two of these of types of signals have proved to be very informative.
A typical example of an application of flow cytometry is the measurement of the contents of individual cells of different components such as DNA and protein. The cells are stained for this purpose with a fluorescent dye which binds specifically and quantitively to the respective component. Each cell which passes through the excitation focus of the instrument thus produces a pulse of fluorescence. This light is collected by appropriate optics and recorded by a light detector. The electrical impulses from this detector, which are proportional with the amount of dye of the cells and thus proportional with the content of the respective cell component, are measured and stored according to their size in an electronic memory--a so-called multichannel pulse height analyzer. In this way it is possible to measure several thousand cells per second with high precision. Thus, a histogram showing the number of cells as a function of their content of the respective component is accumulated.
The excitation light scattered by the cell as it passes through the focus of the instrument may be recorded by a second detector. Depending on the scattering angle the signal carries information about the cross-section or volume of the cell as well as its structure and density.
In some flow cytometers the excitation light is produced by a laser. The fluid flow which carries the particles passes through the laser beam. The flourescence and light scattering of the particles are detected through two separate optical systems situated laterally of the laser beam. In certain types of such flow cytophotometers the flow is a jet in air. In other instruments the fluid flow passes through a closed chamber.
In such flow cytometers the light scattering may be measured both at large and small scattering angles by detectors situated at different agles to the laser beam. For particles with sizes and refractive index similar to that of biological cells (diameter 1-30 .mu.m) the light scattering at small scattering angles will be dominated by refraction from the surface of the particles and may thus be taken as a measure of the size of the particles. The light scattering at angles larger than about
REFERENCES:
patent: 3563660 (1971-02-01), Soloway et al.
patent: 4188121 (1980-02-01), Hirleman et al.
patent: 4286876 (1981-09-01), Hogg et al.
Steen et al, "Differential of Light-Scattering Detection in an Arc-Lamp-Based Epi-Illumination Flow Cytometer", Cytometry 6 (1985), pp. 281-285.
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