Radiant energy – Luminophor irradiation
Reexamination Certificate
2000-08-01
2003-06-17
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
Reexamination Certificate
active
06580081
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
The invention is directed to an arrangement for the detection of fluorescence radiation of matrix-shaped specimen carriers, in particular for the analysis of chemical and biological specimen carriers such as nanotiter plates or biochips.
b) Description of the Related Art
The use of microtiter plates, as they are called, and associated handling technology is an established technique (pharmacological research, clinical practice, etc.) for tasks relating to biotechnical analysis (screening) of large quantities of specimens, preferably for gene analysis (e.g., for diagnosing viruses). This technique is distinguished by the feature that, depending on the embodiment form, 96 (commonest type), 384 or 1536 different specimen substances can be accommodated in a microtiter plate having dimensions of 8 cm×12 cm. Depending on the type of microtiter plate, specimen quantities in the order of magnitude of 100 &mgr;l are required to fill the individual cavities or wells.
By way of increasing effectiveness, research and development work is in progress internationally for the purpose of substantially increasing the quantity of (simultaneously usable) wells while at the same time substantially reducing the required specimen quantities and substantially increasing the specimen throughput. These aims are to be achieved by a transition from microtiter plates to biochips (produced, e.g., with microphotolithography techniques) and fast readout and processing (high throughput screen, HTS) of the biochips.
In order to read out biochips (as well as microtiter plates or any other chemical specimen carriers), the specimen material is irradiated with light in the UV to NIR range, depending on the type of specimen, to cause stimulated radiation in determined substances in the specimens (fluorescent markers are preferably added to the specimen material) due to the effect of the illumination radiation and thereby to detect the presence of determined substances (or components with which a marker substance has coupled) and to determine the proportion thereof in the specimen material.
An individual biochip can contain several tens of thousands of spots (comparable to the wells of the microtiter plate) on a surface of several mm
2
to cm
2
, wherein only specimen quantities on the order of several nanoliters (nl) are required over the sum of all spots. Due to the number of specimens to be evaluated which has accordingly increased enormously, the camera principle, as it is called, has gained increasing popularity for fast readout of the matrix-shaped arrangement of the pixels, aside from the equally well-established scanner principle (with serial laser illumination of the spots and detection of the excited radiation by means of an individual receiver, e.g., a photon counter [SEV or PMT]). In the camera principle, the pixels of the biochip are illuminated in parallel and many or all pixels of the specimen carrier are read out simultaneously using an optoelectronic receiver matrix (e.g., CCD). The following devices make use of the camera principle, for example:
DIANA (Raytest, USA)
ARTHUR fluoroimager (EG&G Wallac, FI)
ArrayWoRx (Applied Precision, USA).
An example for the application of the camera principle is a nanotiter plate readout system from a BMBF joint project, LINDAU (laser-induced fluorescence detection on microstructured specimen carriers for analysis in environmental metrology), which is described in the technical article “Optical Microsystems for Environmental Metrology” (Laser und Optoelektronik, 30 (1), 1998, pages 33-35). According to this publication, direct incident illumination is used via a dichroic mirror for extensive separation of the radiation wavelengths of illumination and fluorescence radiation. Although the type of illumination used here (which is also generally recommended) in the analysis of fluorescence radiation of an object is incident illumination, since it causes the fewest problems with differing transmission of the examined specimens, reflected or scattered components of the illumination light have a noticeable influence on the measurement results—due to the specific surface of the biochip pixels as will be described more exactly in the following, even when using good blocking filters for the wavelengths of the illumination radiation—because the receiver must have a very high sensitivity in view of the much weaker fluorescence radiation.
Another common step in the prior art for increasing sensitivity of detection of fluorescence radiation is the optical imaging of specimen pixels on the receiver by means of high-aperture objectives in order to concentrate as much of the fluorescent light as possible on the receiver, this fluorescent light being weak in itself. For example, suppliers of biochip analysis instruments, e.g., the suppliers of laser scanning systems, General Scanning, Inc. (USA), point to the high aperture of the utilized objective as a selling point. Also, according to general expert opinion, as shown in O. Beyer, “Handbuch der Mikroskopie [Handbook of Microscopy]”, (Verlag Technik Berlin, third printing, 1988, pages 221 ff.) for fluorescence microscopy, with objectives of like magnification those having the higher aperture are preferred, wherein objective immersion is suggested for a further aperture increase.
The surface of every individual spot of a biochip (after a large number of technical steps for preparing the biochip) is generally not flat in an optical sense because it initially has a curved teardrop shape which gradually dries up over the course of processing steps and accordingly takes on an uneven (wrinkled) surface. Accordingly, with the usual incident illumination, problems result in the evaluation channel in that unwanted components of the illumination radiation reach the receiver because of reflections and scattering at the mostly uneven, rough surface. This fact is taken into account in the above-mentioned fluoroimager by EG&G Wallac (see, e.g., company brochure 1442-960-01 (April 1998) for ARTHUR multi-wavelength fluoroimager) to the extent that transmitted light illumination, indirect and lateral incident illumination are offered. A xenon radiator emitting a continuous spectrum with relatively uniform intensity and a UV radiator which is distinguished by intensive discrete spectral lines in the near UV and visible spectrum are used as radiation sources for lateral excitation of fluorescence, wherein the desired illumination wavelength can be selected by choosing an appropriate excitation filter. However, it can not be gathered from the publication whether or not, or to what extent, the quantity of generated fluorescence radiation of different specimens can be compared by means of these types of illumination.
OBJECT AND SUMMARY OF THE INVENTION
It is the primary object of the invention to find a new possibility for detection of fluorescence radiation of matrix-shaped specimen carriers with a large number of individual specimens which permits a highly sensitive quantitative readout of the fluorescence radiation which is characteristically influenced by the individual specimen substances. Another object of the invention consists in making the excitation intensity comparable when using different fluorescing substances.
In an arrangement for the detection of fluorescence radiation of matrix-shaped specimen carriers with a large number of individual specimens presenting metrically ordered pixels on a substance and emitting a fluorescence radiation that is characteristically influenced by the respective specimen substance, with an illumination device for simultaneous excitation of the fluorescence radiation of a large number of substrate pixels, containing a light source and a spectrally narrow-band excitation filter which can be exchanged depending on the fluorescing substance, with transmitting optics for transmitting the fluorescence radiation emitted by individual substrate pixels to a receiver with a large number of receiver elements and an exchangeable filter in front
Jena-Optronik GmbH
Reed Smith LLP
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