Optical: systems and elements – Lens – With viewed object or viewed field illumination
Reexamination Certificate
2000-11-15
2002-05-07
Epps, Georgia (Department: 2873)
Optical: systems and elements
Lens
With viewed object or viewed field illumination
C359S809000, C359S599000
Reexamination Certificate
active
06384989
ABSTRACT:
This invention relates to an optical system with a magnifying objective and an incident-light illumination which is intended to image the surface, provided with an analysis preparation, of the bottom of a well of a microtiter plate made of a transparent material.
BACKGROUND OF THE INVENTION
Optical systems of the type referred to are utilized in particular for the evaluation of ELISPOT (Enzyme Linked Immuno Spot) assays. This method has been devised for identifying beta cells secreting individual antibodies. It finds application also in the identification of cytokine secretions of special subgroups of leukocytes or T-lymphocytes from peripheral blood as well as of monocytes and granulocytes. In the ELISPOT method proteins secreted from the cells are identified with antibodies, and point-shaped stains referred to as spots are produced by means of special coloring methods, which spots are permanent and suitable for evaluation, both visually and by means of image analysis. In laboratory routine, ELISPOT assays are typically performed using microtiter plates (MTP), also commercially designated as filter plates or multiscreen plates. The microliter plates comprise small interlinked cups also referred to as cavities or wells. The bottom of the cups is conventionally formed by a filter membrane to which the point-shaped objects subject to the assay adhere. A standard size MTP comprises 96 wells.
To evaluate an ELISPOT assay it is necessary to count the small spots of up to 1,500 per well which are visible on the bottom of the wells of an MTP, the bottom providing the measurement surface. To this effect, automatically operating evaluating devices are used which image the surface of the bottom of the individual wells with an about twenty- to fortyfold magnification, take the images with an electronic camera, and evaluate the digitized image data by means of an electronic computer.
The processes of imaging and evaluating the microtiter plates present the problem that reflection outside the filter membrane produces a phantom image of the filter membrane, so that the spots occurring in the marginal area are detected twice, causing errors to be introduced in the measurement process. To prevent this from happening, one possibility would include masking the areas lying outside the well with a circular mask. This would however necessitate an exact positioning of the individual wells of the microtiter plate relative to the optical axis, which is a highly complex and difficult task considering that the microtiter plate manufacturers do not guarantee an absolutely accurate positioning of the filter membranes and the adjustment accuracy of an X-Y table necessary for positioning would have to lie in the range of 2 &mgr;m, approximately. To solve this problem, one proposal hitherto made includes detaching the filter membranes from the MTP, adhering them to a film and then optically detecting and evaluating the adhered filter membranes. Separating the filter membranes from the MTP prevents the generation of a phantom image by reflection. The disadvantages of this approach are however that the processes of detaching and adhering require an additional operation, that individual membranes may shift out of place as the filter membranes are detached, and that the film materials service life is limited. On the other hand, recent image analytical devices provide an easy way to determine the position of the adhered filter membranes and to cause an X-Y table to travel to the center of the image, if and when necessary.
In another known system of the type referred to, the positioning of the magnifying objective initially involves detecting the MTP from a major distance without appreciable magnification and then using this image for measuring and storing the exact position of the individual wells. By means of the stored values it is then possible to position the magnifying objective above the individual wells automatically. This necessitates a relatively high construction effort for the optical system and the positioning table, and additional memory capacity is needed for storing the position-related data.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an optical system of the type initially referred to which enables the measurement surfaces of a transparent MTP to be imaged without the occurrence of reflections interfering with the image evaluation.
According to the present invention, this object is accomplished in that the incident-light illumination is formed by an annular diffuse light source arranged concentrically with the optical axis, and that on the rear side of the microtiter plate provision is made for a powerful, approximately point-shaped light source which is located centrally relative to the optical axis.
By virtue of the illumination according to the invention the MTP area lying outside the measurement surface of the filter membrane is imaged completely white. By contrast, the prepared measurement surface is set off against this light background in a lightly gray color. Reflections and material interferences in the area lying outside the measurement surface needing to be evaluated are no longer visible. This effect can be further enhanced by arranging the approximately point-shaped light source behind a diffuser disk, thus accentuating the contrast between measurement surface and background.
The optical system of the present invention has the advantage of enabling fault-free imaging and evaluation of the measurement surfaces of an MTP's filter membranes. Even minor inaccuracies in locating the measurement surface centrally to the optical axis will not impair the accuracy of measurement so that the demands placed on the positioning accuracy of the table carrying the MTP and movable in the X and Y direction may be less high, accordingly enabling the table to be manufactured more economically. The distinct contrast of the measurement surfaces from the light background surrounding them furthermore makes it possible to determine the position of the measurement surfaces by means of image analysis and to move them automatically to the image center under appropriate table control. An automatic evaluation of microtiter plates is hence readily possible.
The present invention will be described in more detail in the following with reference to an embodiment illustrated in the accompanying drawings. In the drawings,
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patent: 5267087 (1993-11-01), Wiedermann
patent: 5752767 (1998-05-01), Muehlemann
patent: 5781338 (1998-07-01), Kapitza et al.
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Collard & Roe P.C.
Seyrafi Saeed H
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