Radiant energy – Luminophor irradiation
Reexamination Certificate
1999-01-25
2001-01-30
Evans, F L (Department: 2877)
Radiant energy
Luminophor irradiation
C250S36100C
Reexamination Certificate
active
06180948
ABSTRACT:
The present invention concerns an image recording system for the image recording and evaluation of analytical test elements comprising a holding unit for test elements, a lens system which forms an image of a test element in the holding unit on a CCD chip, a CCD chip, which records an image of the test element and passes it on to an evaluation unit as well as an evaluation unit which converts the signals of the CCD chip into a representation of an image of the test element wherein the focus of the lens system has a fixed setting and the lens system is a reducing optical system with an aperture of at least 0.7 on the image side; the distance between the CCD chip and the nearest lens of the lens system is less than 15 mm and this distance is held constant to at least 10 &mgr;m.
The present invention relates in particular to the field of molecular biology in which biomolecules with a label or dye are detected and their position is determined on a usually flat analytical carrier.
Biomolecules within the sense of the invention are in particular nucleic acids, nucleic acid fragments and proteins.
The labelling and the detection of nucleic acids and proteins is a standard method which is carried out in most biochemical, biological or biomedical laboratories.
Biomolecules can in practice be directly labelled in three different ways:
1. by radioisotopes
2. by fluorescent dyes
3. by colloidal gold.
However, an indirect detection is usually carried out due to the higher sensitivity by labelling the biomolecules with a hapten or biotin and then subsequently detecting by means of antibodies or streptavidin.
The detection proteins (antibody or streptavidin) are then usually coupled to enzymes such as alkaline phosphatase or horseradish peroxidase which catalyse a colour or chemiluminescence reaction via suitable substrates in order to achieve higher detection sensitivities. These reactions are ultimately used for the detection.
Even nowadays radioactive methods are still used considerably more than non-radioactive methods in the field of nucleic acid labelling although they have the disadvantages associated with radioactive isotopes. The major disadvantages are the short half-life of the isotopes as well as the health and environmental hazards of radioactive isotopes. For this reason alternative methods without radioactive isotopes were developed for the labelling of biomolecules. One possibility is to directly couple biomolecules to fluorescent dyes which can then be detected by fluorescence microscopy. However, so-called blotting methods (Southern, Northern and dot blots for nucleic acids and Western blots for proteins) are common in the field of biochemistry. Fluorescent dyes are usually not sufficiently sensitive for these areas of application since problems occur with the signal background and the intrinsic fluorescence of the blotting membranes used. However, a non-radioactive detection is possible on such membranes by direct or indirect binding of the proteins to enzymes such as alkaline phosphatase or peroxidase. Such enzymes catalyse an emission of chemiluminescence light when suitable substrates are added. Similarly nucleic acids can be provided with haptens such as e.g. digoxigenin or biotin and these labels can be detected with specific antibodies which are in turn bound to enzymes.
In methods in which a dye is detected directly, an image of the membrane can be evaluated visually or photographed with standard devices. However, a chemiluminescence reaction is preferably used for the detection processes described above since it is very rapid and very sensitive. It has turned out that a detection by means of a chemiluminescence reaction is at least as sensitive as radioactive detections but can be carried out much more rapidly. At present chemiluminescence signals are recorded by placing an X-ray film on membranes. X-ray films have the advantage that they have a very good resolution and relatively large objects e.g. blots with a size of 40×30 cm can be evaluated. However, a disadvantage of X-ray films is their small dynamic range which is about 2 orders of magnitude and hence is less suitable for distinguishing between strong and weak signals on the same film. For this reason numerous exposures of the same analytical test element are necessary if a quantification of the signals is desired. A further disadvantage of X-ray films is that a special laboratory as well as special chemicals are necessary to develop them and the chemicals have to be disposed of after use.
So-called phosphorus imagers are known in the prior art some of which also record luminescence images. However, the phosphorus imagers are disadvantageous due to their high price as well as the relatively small dynamic range and the low spatial resolution.
Furthermore cooled CCD cameras are on offer in the market for the evaluation of luminescence reactions. However, known instruments have the disadvantage that different optics have to be used for the various sizes of objects that are to be evaluated. In addition it is necessary to focus the optical system in these instruments. Since in biochemical laboratories a large number of analytical test elements such as blots and gels are evaluated, focussing is time-consuming and inconvenient for the operator.
The object of the present invention was to provide a system for evaluating analytical test elements which gives a high sensitivity with an adequate dynamic range and also enables the evaluation of different test elements without requiring a focussing.
The object mentioned above was achieved by an image recording system for the evaluation of analytical test elements. In particular it was found that the object can be achieved when a special lens system is selected which, on the image side, has a high aperture and a small distance from the sensor. However, this is associated on the image side with a very small depth of focus. On the object side a lens system according to the invention has a large depth of focus by which means it is possible to avoid focussing if a holding unit for test elements is present which essentially fixes the distance between the test element and lens system. Due to the large depth of focus on the object side of the lens system used, variations in the distance between the lens system and test element due to variations in the thickness of the test element are insignificant. The problem associated with the lens system according to the invention that the depth of focus is very small on the image side was solved according to the invention by using a compensator which compensates for variations in the distance between the lens system and CCD chip. A further advantage of the system according to the invention is a simplification of the quantitative evaluation by a constant image scale.
The image recording system according to the invention serves to record images of and evaluate analytical test elements. Such test elements are for example membranes on which blotting reactions are carried out or also gels such as those that are for example used for DNA sequencing. However, those test elements are also suitable according to the invention on which fluorescent or dyed analytes are located. The lens system used in the image recording system is designed so that the advantages are achieved particularly for test elements with a size above ca. 4×4 cm.
The holding unit for flat test elements can for example be a plate on which the test elements are placed. However, the holding unit is preferably in the form of a drawer on the bottom surface of which the test elements are placed and pushed into the image recording system together with the drawer. A holding unit can for example also be composed of guide grooves into which the test element, optionally on a carrier, is inserted. According to the invention the function of the holding unit is to arrange the test elements at an essentially constant distance from the lens system in such a manner that the examination area of the test element is arranged perpendicularly to the optical axis of the lens system and that
Gutekunst Martin
Holtke Hans-Joachim
Arent Fox Kintner & Plotkin & Kahn, PLLC
Boehringer Mannheim GmbH
Evans F L
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