Optics: measuring and testing – Sample – specimen – or standard holder or support
Reexamination Certificate
1998-10-13
2001-02-20
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Sample, specimen, or standard holder or support
C356S422000
Reexamination Certificate
active
06191852
ABSTRACT:
The invention is based on a measurement system for detecting optical signals of microassays, in which the signal-generating test objects are arranged on an investigation surface of a planar carrier, comprising an optical imaging arrangements which reduces the size of the test objects to be measured in such a way, that all the objects are imaged completely on a two-dimensional, photosensitive image sensor. The optical signals are converted by the image sensor into electronic image signals, which are evaluated by a measurement computer in a known manner and processed further.
“Test objects” in the context of the invention are to be understood to mean fluorescent or luminescent and/or fluorescent- or luminescent-marked samples which are provided on the carrier or in microtitre plates and in which a chemoluminescent or bioluminescent reaction proceeds in the case of luminescence on account of molecular interactions, in which reaction photons can be liberated and detected, or fluorescence arises in the case of fluorescence on account of the interaction of a fluorescent die with which the objects are marked, given irradiation by suitable excitation energy, with the result that photons can be liberated and detected. The samples themselves may be present in the form of dissolved chemical components or else in the form of biological test systems, such as for instance, in the case of enzymatic reactions, antigen-antibody couplings, protein binding assays, ligands-receptor interactions or receptor assays. In this case, the biological test system may be configured as a cellular assay (adherent or suspension cells, primarily mammalian cells, but also plant cells, bacteria, fungi, yeasts or viruses) or else may comprise subcellular constituents, such as e.g. isolated cell nuclei or cytoplasm agglomerates, or else may comprise artificial carriers, such as e.g. plastic beads or glass microspheres, on which biologically active material, generally cellular or subcellular constituents, has been applied, an optical signal in the form of photons being liberated as a result of the interaction of different components.
A problem that frequently arises in the measurement of luminescence or fluorescence in biomedical assays is, that the optical signals correlated with the biological interaction are generally small enough that bioluminescent or biofluorescent events can usually be detected only using photomultipliers (light intensifiers). In the limit range of detectability, it is necessary to use luminescence measurement systems (photon counting systems).
If the intention is for e.g. microtitre plates (MTP) having dimensions of approximately 130 mm×86 mm, depth approximately 10-14 mm, which contain 96, 384 or 1536 test holes, to be optically measured simultaneously using imaging methods, then two-dimensional luminescence measurement systems are necessary for this. The prior art in this context is to image the MTP by means of an optical lens arrangement onto the photocathode of an image intensifier (entry window) and to amplify the impinging photons after photoelectric conversion as photoelectrons in a microchannel plate (MCP=multichannel plate). At the exit window of the MCP, the multiplied electrons impinge on a luminescent phosphor, where they engender, in a spatially resolved manner, a light signal which is amplified by up to 1,000,000 times relative to the input and can be detected, in a spatially resolved manner, using a CCD sensor.
An output image, that has been intensified in such a way, can be evaluated with the aid of image-processing processes, the brightness in each hole of a microtitre plate being calculated as the number of recorded photon events. Corresponding systems are commercially available as so-called MTP readers from various companies. If the intensities are sufficiently high and the integration time is unimportant, it is also possible to have recourse to commercially available cooled CCD systems instead of the image intensifier.
By comparing different luminescence measurement systems from various manufacturers, it was possible to demonstrate that photons, which generate an electron on the photocathode, can be detected by the known luminescence measurement systems having single- or two-stage amplification. A higher level of amplification after photoelectric conversion, and that is to say downstream of the photocathode, does not lead to an increase in the system sensitivity.
An increase in sensitivity on the basis of a higher quantum yield of the photocathode of an image intensifier is theoretically possible. However, physical limits and the absence of corresponding commercial detectors afford no technically realisable solution at the present time.
In order to image an object, the microtitre plate in the case described here, onto the photocathode of an image intensifier, all the manufacturers use an objective of high light-gathering power. Some manufacturers use standard photo objectives, others use specially corrected objectives having a high f-number. The best high-performance objectives used to date already have a very high light intensity with an aperture ratio of approximately 1:1.0 and a focal length of 50 mm. An optical arrangement having a significantly higher light-gathering power cannot be constructed for physical reasons.
Owing to the three-dimensional nature of the microtitre plate with its holes having a depth of approximately 10-14 mm, in the case of conventional optical imaging it is necessary to observe an object distance of approximately 70 cm (distance: MTP from image sensor) owing to the geometric vignetting that occurs at the edge holes. A housing in which the microtitre plate and the detection system are integrated in a completely light-tight manner for the luminescence measurement system must be correspondingly high. This geometrical distance r between light origination and detector has a particular disadvantageous effect with regard to the system sensitivity since the intensity decreases at I/r
2
. The light quantum originating statistically in a hole of the microtitre plate leaves the hole diffusely, with the result that it is possible to detect, purely geometrically, only a fraction (surface of the hemisphere with a radius of 70 cm in relation to the aperture of the objective with a diameter of 5 cm) of a few thousandths. No fundamental improvement in the system sensitivity can be achieved with conventional optical imaging arrangements such as lens systems or else mirror systems.
In order to ensure a high sample throughput in an acceptable time in the event of screening biological test systems with optical signal processing during a test of a few 100,000 substances, it is necessary to resort to imaging methods owing to the advantage of parallel processing, e.g. in microtitre plates. Owing to the low luminous intensity of the bioluminescent or biofluorescent reactions, integration times of a few minutes are frequently necessary for a statistically secure signal
oise ratio in the photon counting mode.
In order to increase e.g. the capacity of a robot installation for the investigation of luminescence or fluorescence signals in microtitre plates by reducing the integration time for the measurement of microtitre plates and/or to reduce e.g. the number of cells per test hole and/or to reduce the size of expensive substrate quantities for an enzyme reaction, the object consisted in increasing the sensitivity of the known luminescence or fluorescence measurement systems.
In the case of an optical measurement system having an optical imaging arrangement for the test objects to be measured, which test objects are situated on the investigation surface of the carrier, and a two-dimensional, photosensitive image sensor onto which all the objects are completely imaged, this object is achieved according to the invention by the fact that the optical imaging arrangement comprises a high-resolution glass-fibre taper element having a large-area and a small-area end and the end surfaces are chosen such that the large-area end surface corresponds at least to the
Bechem Martin
Paffhausen Wolfgang
Bayer Aktiengesellschaft
Font Frank G.
Norris McLaughlin & Marcus P.A.
Ratliff Reginald A.
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