Optics: measuring and testing – By shade or color – With color transmitting filter
Reexamination Certificate
2001-01-10
2003-08-05
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By shade or color
With color transmitting filter
C250S458100
Reexamination Certificate
active
06603553
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from a previously filed German application DE 100 03 754.2 filed on Jan. 28, 2000, which application is herein incorporated by reference.
BACKGROUND OF INVENTION
The invention relates to an arrangement for detecting fluorescent light from a plurality of specimen points, in particular from microgene spots or microbiospots, the specimen points being arranged on a slide, having at least one light source for simultaneously illuminating the specimen points with excitation light, and having detection means, comprising detection elements, for simultaneously detecting the fluorescent light from the individual specimen points.
Such arrangements are used, for example, for investigations in molecular biology or gene technology. The specimen to be investigated is mounted for this purpose on a slide and brought into contact with a fluorescing label. The substances present in the specimen and having an affinity with the label bind the label and can therefore be excited to emit fluorescent light. Conclusions on the composition of the specimen can therefore be drawn by detecting the fluorescent light emitted by a specimen.
European Patent Application EP 0 947 823 A2 describes a device for detecting a fluorescent dye in specimens, the individual specimen points being arranged on a slide. This slide is designed in the form of an optical conductor. Light is launched into the slide in order to excite the fluorescent dye at the individual specimen points. The fluorescent dye is then excited by the evanescent field formed at the interface between the slide and the specimen points. The known device can be used to detect simultaneously the fluorescent light from a series of specimen points, for which purpose, for example, a linear photodiode array, or else a CCD row is used as detection device. In addition to statistical investigations, in the case of which there is no need to assign the detected fluorescent radiation to a specific specimen point, the device described in EP 0 947 823 A2 also permits simultaneous individual investigations of a multiplicity of specimen points in the case of which the fluorescent light from each individual specimen point is detected with the capability of assignment. For this purpose, it is proposed to use a lens system with the aid of which the emitted fluorescent radiation of the individual specimen points can be focussed onto the device for detecting the fluorescent radiation.
In practice, both as regards design and with regard to the conduct of measurement, this measure turns out to be very expensive. Even, the implementation of a suitable lens system requires high precision in fabrication. In addition, the lens system not only has to be arranged, but also very precisely adjusted, between the slide, provided with specimen points, and the detection device, in order to achieve the desired focussing effect.
SUMMARY OF INVENTION
The present invention now proposes a very simple but extremely effective measure to prevent superimposition of the fluorescent light emitted by a plurality of specimen points at the location of a detection element, that is to say at the location of the measured value detection.
According to the invention, it is proposed for this purpose to select the spacing between the specimen points and the respectively assigned detection elements to be as small as possible so that the fluorescent light from the individual specimen points is detected in the manner of the production of a photographic contact print. Specifically, according to the invention it has been recognized that it is possible in this way to achieve very good results very easily.
In an advantageous way, the detection means could comprise a dedicated detection element for each specimen point. Depending on requirement, it would be possible as an alternative to this for each specimen point to be respectively assigned a plurality of detection elements. The respective application is the determining factor in this case.
In order to avoid superimposition of the fluorescent light from a plurality of specimen points at the location of a detection element, or of a plurality of respectively assigned detection elements, it is also possible to provide additional means within the scope of the present invention. In an advantageous variant, arranged for this purpose between the specimen points and the detection elements is at least one element which is transparent to fluorescent light emitted by a specimen point essentially in the direction of the assigned detection element, and totally reflects the fluorescent light emitted in the direction of adjacent specimen points, such that this fluorescent light is not detected. Such an element can be implemented, for example, in the form of a glass plate which is arranged between the specimen points and the detection elements, provided that the slide is likewise of plate-shaped design and the detection elements are arranged in a plane parallel to the slide and opposite the surface of the slide which is provided with the specimen points. The fluorescent light emitted at a steep angle by the specimen points in the direction of the detection elements can penetrate such a glass plate without hindrance, whereas the fluorescent light emitted in the direction of adjacent specimen points is totally reflected at the glass/air interface and deflected once more in the direction of the slide. By contrast with the lens system described in EP 0 947 823 A2, such a glass plate has the advantage that it need not be specially adjusted. Moreover, it is also less expensive to produce and maintain.
As a supplement, or also alternatively to the element described above, it is also possible effectively to suppress superimposition of the fluorescent light from a plurality of specimen points at the location of a detection element or a plurality of respectively assigned detection elements with the aid of mechanical, opaque barriers which are arranged between the specimen points and the detection elements such that they delimit detection channels for the individual specimen points and assigned detection elements.
As already mentioned, the detection means of the arrangement according to the invention comprise a dedicated detection element for each specimen point. In this context, the use of photodiode arrays or CCD chips has proved itself in practice.
The illumination of the specimen points with excitation light can also be implemented in various ways within the scope of the arrangement according to the invention for detecting fluorescent light from a plurality of specimen points. An advantageous variant is so-called transillumination. In this case, the slide is transparent at least to the excitation light. The light source is then arranged on the side of the slide averted from the specimen points such that the specimen points are illuminated through the slide. Another advantageous variant is total internal illumination by the slide. The slide is designed in this case in the form of an optical conductor. Moreover, means are provided for launching the light of the light source into the optical conductor such that the specimen points are illuminated by the evanescent field formed at the interface between slide and specimen points.
Finally, it may be mentioned further that in a particularly advantageous variant of the arrangement according to the invention for detecting fluorescent light from a plurality of specimen points, a filter arrangement which is essentially transparent only to the fluorescent light to be detected is also additionally arranged between the specimen points and the detection elements. In a simplest embodiment, such a filter arrangement could comprise only a blocking filter for the excitation light emitted by the light source, in order to suppress interference caused thereby when detecting the fluorescent light emitted by the specimen points. For the case in which the specimen points have been brought into contact with various labels before measurement, resulting in fluorescent light of different wavele
Evans F. L.
Houston Eliseeva LLP
Leica Microsystems Heidelberg GmbH
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