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 cmxc3x9712 cm. Depending on the type of microtiter plate, specimen quantities in the order of magnitude of 100 xcexcl 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 mm2 to cm2, 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 (EGandG 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 xe2x80x9cOptical Microsystems for Environmental Metrologyxe2x80x9d (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 resultsxe2x80x94due 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 radiationxe2x80x94because 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, xe2x80x9cHandbuch der Mikroskopie [Handbook of Microscopy]xe2x80x9d, (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 EGandG 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.
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 of the receiver for passing wavelengths of the fluorescing substance and blocking excitation wavelengths, the object stated above is met according to the invention in that the transmitting optics contain an imaging objective by which every substrate pixel is correlated to a determined group of receiver elements of the receiver and which is outfitted with an additional aperture stop for limiting the angular area of the fluorescence radiation detected by the objective, so that the receiver elements which are correlated respectively with a determined substrate pixel receive substantially no fluorescent light of neighboring substrate pixels, and in that the illumination device has a darkfield illumination unit for large-area illumination of a large number of substrate pixels by formation of an excitation beam bundle which is symmetric with respect to the axis of the objective and which cuts out the aperture of the objective as well as a substantial angular area surrounding the objective aperture.
A micro-objective is advantageously used as the objective in order to obtain a sufficiently magnified imaging with few imaging errors of the substrate pixels on the receiver. To prevent detectable crosstalk of the fluorescence radiation of neighboring substrate pixels on receiver elements not assigned to them, the additional aperture stop is preferably dimensioned in such a way that the effective numerical aperture of the micro-objective is reduced by 10 to 30%.
A micro-objective with an aperture stop plane located outside of the objective is advisably provided as objective, wherein the additional aperture stop can be arranged in this aperture stop plane in a simple manner.
A micro-objective with ten-times magnification and an aperture of 0.2 to 0.25 can advantageously be used. In this case, the effective aperture of the micro-objective is reduced by 15 to 30% by the additional aperture stop.
In a micro-objective which images to infinity, the transmitting optics are advisably supplemented by a tube lens arranged in a tube which is adjustable in length for generating a sharp optical imaging of the substrate pixels emitting the fluorescence radiation on the associated groups of receiver elements.
The darkfield illumination unit is advisably constructed symmetrically for reasons of homogeneity of the excitation radiation in the substrate plane. This can be carried out advantageously by means of light guides whose light outlet windows, which are distributed about the optical axis of the objective, are focused on a spot of the substrate. Also, a rotationally symmetric darkfield condenser for generating a ring-shaped excitation beam bundle is preferably used as darkfield illumination unit for transmitted light illumination of the substrate. A dry darkfield condenser is particularly suitable for this purpose. When a darkfield condenser is used it is advantageous to arrange additional optics in front of it as a collector.
In order to make fluorescence radiation measurements comparable when exciting with different excitation wavelengths, the spectral emission of the illumination device and the spectral sensitivity of the receiver are to be adapted to one another in such a way that their product gives approximately a constant over a wavelength range required for the fluorescence substances to be detected. For optimum excitation of fluorescence radiation of different fluorescing substances, an intensive continuous light source with an exchangeable bandpass filter adapted to the excitation wavelength of the fluorescing substance is advantageously used.
A halogen lamp or xenon lamp is advisably used as a broad-band light source. The light source is advantageously coupled with the darkfield illumination unit via a light guide so that the light source is separated spatially, and therefore preferably thermally, from the rest of the components. This coupling is preferably carried out by means of a liquid light-conducting cable in order to minimize transmission loss in the intensity of the excitation radiation in the desired wavelength range.
When using a light source coupling via light-conducting cable, the spectral emission of the light source, the spectral transmission of the light guide and the spectral sensitivity of the receiver are adapted to one another in such a way that the product of these three spectral quantities gives approximately a constant.
The optical subassemblies mentioned above are advantageously comprised in exchangeable modules for adapting to specific cases of application. The arrangement according to the invention is advisably divided into, successively, a light source module, preferably with connected light guides for transmitting the excitation light, a module for coupling in light and beam expansion, a substrate carrier module, an imaging module for the fluorescence radiation and a camera module, wherein the substrate carrier module in particular contains a substrate displacement unit for recording large-area substrates and for continuously successive processing of a plurality of substrates.
The basic idea of the invention is based on the insight that the recommendation applied for fluorescence analysis in the prior art, to realize the transmission of the very weak fluorescent light onto the receiver with objectives having the highest possible aperture, is not favorable in every case. As has been shown by theoretical and empirical results in trials with the camera principle, large aperture values of real imaging optics in a quantitative detection of fluorescence intensities of small and closely adjacent matrix elements lead to noticeable crosstalk (when imaging on a CCD matrix, components of the fluorescence radiation of a determined substrate pixel also reach neighboring receiver elements which should actually detect exclusively the fluorescence intensity of a neighboring substrate pixel).
In particular with a high objective aperture, the crosstalk causes the fluorescence measurements to be influenced in such a way that this falsifying of the individual specimen measurement values cannot be tolerated for quantitative fluorescence analysis. This problem is solved according to the invention in that the given aperture of a magnifying imaging objective is limited to such an extent that the crosstalk of the fluorescence radiation is sufficiently suppressed. In this respect, the extent to which the aperture is reduced depends essentially on the correction factor of the objective relative to imaging errors (aberrations). Accordingly, with the use of micro-objectives (which are generally easily corrected) reductions of less than 30% are entirely adequate, whereas in the case of photographic objectives aperture reductions of up to 50%, sometimes even up to 65%, are necessary for sufficient prevention of crosstalk.
It is not only in the latter case that a reduction in the aperture also entails a considerable light attenuation of the fluorescence radiation that must be compensated by suitable steps in the illumination of the substrates. In addition, the various fluorescing substances that are used are taken into account with respect to their specific optimum excitation wavelength and a uniformly intensive excitation with the different excitation wavelengths through the use of a suitable continuously intensive light source, transmitting media with correspondingly high transmission and matched spectral characteristics of the illumination and the receiver. For the last step, the spectral emission of the illumination device and the spectral sensitivity of the receiver are adapted to one another in such a way that their product gives approximately a constant over a wavelength range required for the fluorescing substances that are to be detected. It must be mentioned in this connection thatxe2x80x94strictly speakingxe2x80x94the product of the spectral emission of the illumination on the excitation wavelength and the spectral sensitivity of the receiver on the emission wavelength of the fluorescence would have to give a constant. However, since the excitation wavelength and the fluorescence wavelengths of the usual fluorescent dyes are only separated by several tens of nm, this slight imprecision can be disregarded when the product of the spectral characteristics is sufficiently constant over the entire necessary wavelength range.
Accordingly, it is possible with the arrangement according to the invention to minimize the crosstalk of the fluorescence radiation of neighboring substrate pixels on the receiver and to compensate for the consequent inevitable deficit in the detected fluorescence intensity by more efficient illumination of the examined substrate pixels. Accordingly, a quantitative readout of the fluorescence radiation is realized in which every individual specimen substance on a specimen carrier with a large number of individual specimens can be analyzed in a highly sensitive manner with respect to the quantity of contained fluorescing substance and the results are comparable even when using different fluorescing substances.
The invention will be explained more fully in the following with reference to an embodiment example.