Optics: measuring and testing – For optical fiber or waveguide inspection
Patent
1995-09-18
1997-11-04
Font, Frank G.
Optics: measuring and testing
For optical fiber or waveguide inspection
356 73, G01N 2100
Patent
active
056845758
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to an optical arrangement for flow cytometers.
A flow cytometer is an instrument for measurement of the fluorescence and light scattering of individual biological cells and other types of microscopical particles. In the flow cytometer, the cells are carried by a laminar flow of water through the focus of a high intensity light source. The cells are typically stained with a fluorescent dye which binds specifically to one particular cell constituent. Each cell passing through the focus will thus emit a short pulse of fluorescence and scattered light. The intensity of the fluorescence will be proportional to the cellular content of fluorescent dye and thereby with the cellular content of the stained constituent. The intensity of the scattered light and its angular distribution is a complex function of the size, shape, structure and chemical composition of the cell. By measuring with separate detectors the light scattering at small and large scattering angles, respectively, it is thus possible to distinguish cells on the basis of size, shape, and structure.
For some purposes, the cells may be stained by two or three different dyes which bind to different cellular constituents and fluoresce at different wavelengths. The corresponding spectral components of the fluorescence can be separated by dichroic mirrors and band filters and measured by separate detectors. Hence, each cell may generate several signals; typically two light scattering signals--low and large angle scattering--and two or three fluorescence signals. This technology is well known and has been published in many articles, e.g. in "Flow cytometry and sorting" (Melamed, M. R.; Lindmo, T; Mendelsohn, M. L., Eds.), Wiley-Liss, New York 1990.
The cellular content of the constituent(s) to be measured may be quite small, that is down to about 1.multidot.10.sup.-18 g/cell. The demands on the sensitivity of the instrument are correspondingly high. In order to achieve such sensitivity the excitation light has to be concentrated into a very small and correspondingly intense focus. Furthermore, the optics which collects the fluorescence and scattered light must have the highest possible numerical aperture. It is essential also that any light from other sources than the cells, e.g. the background due to fluorescence and light scattering from optics and other components in the optical path, is as low as possible.
There are two major types of flow cytometers: a) Instruments employing a laser as the source of excitation light, and b) instruments using a high pressure arc lamp with xenon or mercury. The laser-based instruments have the advantage that the excitation light can be focused into a very small and correspondingly intense focus. Furthermore, the beam of excitation light is near parallel, which simplifies the distinction of light scattered to different angles. Arc lamp-based instruments have the advantage that the spectrum of the light source contains all wavelengths from UV through the visible spectrum. Hence, by means of appropriate filters the proper wavelength for excitation of any fluorescent dye can be selected, thus making this type of instruments more versatile.
All laser-based flow cytometers have essentially the same optical configuration, namely so that the vertical sample stream cuts through the focus of a horizontal laser beam and so that this focus is intersected at a 90 degree angle by the optical axis of the optics which collects the fluorescence and the light scattered to large angles, i.e. around 90.degree.. Behind the light collecting optics fluorescence and light scattering are separated by a dichroic mirror and directed onto separate light detectors. The fluorescence may be further split into different spectral components by additional dichroic mirrors and measured by separate detectors.
The light of the focused laser beam is near parallel, that is falling within a light cone of about 2.degree. or less. Hence, the light scattering at low scattering angles is measured through an
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Font Frank G.
Viena Eisenberg Jason D.
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