Various applications of fluorescence techniques to analyze biological samples are known to people skilled in the art. In case of electrophoretic techniques proteins or DNA are labeled with a fluorescence probe to visualize their electrophoretic bands in gels or columns. In addition, most biochip applications so far are based on a fluorescence read-out, whereas the specific binding of a fluorescence-labeled target molecule to a probe molecule immobilized on a solid support is monitored. Applications for DNA analysis in the liquid phase include fluorescence hybridization probes like the double-stranded DNA binding dye SybrGreenI or FRET (Fluorescent Resonance Energy Transfer) probes utilizing two fluorescence probes and energy transfer. A very important application for fluorescence techniques in the liquid phase is the quantification of PCR products in real time, the so-called real-time PCR.
In all these cases, a fluorescence reading device is needed that provides light of a certain wave length to excite the fluorescence label of the assay and that is able to detect the fluorescence light form said label emitted at a somewhat different wavelength. One major problem of all fluorescence reading devices is the enormous intensity of the excitation light in comparison with the fluorescence light emitted by the dye and therefore, one has to assure that the excitation beam does not hit the detector in order to monitor the fluorescence signals accurately. In other words, the optical path of the excitation light has to be different from the optical path of the fluorescence light, at least partially.
The realization of the fluorescence principle is quiet easy, when only one fluorescence probe has to be monitored in the liquid phase of e.g. a capillary. Here, e.g. a white light source together with a set of dichroic mirrors and filters is sufficient to meet the requirements. However, if more than one fluorescence label is present in the sample, a lateral distribution of spots on a solid support or the fluorescence of a microtiter plate has to be monitored, the requirements for the fluorescence reading device are more difficult to fulfill.
In principle, there are two different strategies to excite and monitor the fluorescence of a lateral distribution of sites. The first strategy is to scan the lateral distribution of sites, whereby the individual sites are successively analyzed one at a time. The second strategy is to illuminate the whole distribution of sites simultaneously and to image the corresponding fluorescence e.g. on a CCD chip. The scanning strategy has the obvious drawback that either the support has to be moved in two dimensions (WO 03/069391, DE 102 00 499), the detector has to be moved with respect to the support (US 2002/159057), the detector has to move in one dimension and the support in the other dimension or the optics has to include one or two dimensional scanning means i.e. galvo mirrors. On the other hand, the main difficulty of the strategy to illuminate the whole support simultaneously is to assure a homogeneous illumination across the whole distribution of sites. An alternative to the homogeneous illumination of the whole distribution of sites is the use of an array of light sources, whereby each site is illuminated by its own light source. DE 101 31 687 describes this strategy for the evaluation of PCR in a thermocycler with a plurality of wells using a beam splitter and an array of LEDs for illumination. DE 101 55 142 describes the dark field monitoring of fluorescence signals, wherein the microarray is illuminated by an array of LEDs, too, but in this embodiment no beam splitter is needed.
Concerning the requirement to separate the optical path of the excitation beam and of the fluorescence light at least partially, there are again two different possibilities. The first possibility is the so called epi-illumination, whereby beam splitters are utilized and the excitation beam and the fluorescence light share at least part of the optical train. The second possibility is the use of oblique illumination. Here, the excitation beam is arranged in such a way that it has a certain angle to the normal of the support surface and the corresponding reflection of the excitation beam is outside of the acceptance angle of the detection system (e.g. US 2002/0005493 A1, EP 1 275 954 A2).
US 2003/0011772 A1 describes an optical apparatus to simultaneously observe a plurality of fluorescence dyes in a probe using a beam splitter. DE 197 48 211 A1 discloses a system to monitor the fluorescence signals generated in the wells of a microtiter plate simultaneously using a beam splitter, a field lens and an array of lenses focusing the light into each well. The detection is performed by imaging the light onto an array of photodiodes or a CCD chip. The fluorescence light collected in this embodiment of the system is appointed by the amount of dyes excited by the light cone of the focusing lens and therefore is dependent on the fill level of the well. WO 99/60381 claims an instrument for monitoring PCR reactions simultaneously in a plurality of vials in a temperature cycled block. The optical components of this instrument include again a beam splitter, a field lens, an array of vial lenses focusing individual light beams into each vial and a detection mean focusing the emission light onto e.g. a CCD detector. Due to the necessity of an array of vial lenses, the size and the lateral density of individual sites is limited. The JP 2002014044 describes a fluorometric apparatus to monitor fluorescence generated at a plurality of wells. The optical components comprise a beam splitter and a lens system to illuminate the wells collectively with light being parallel to the direction of the depth of the wells. However, the image forming optical system condenses the light onto a detection mean. U.S. Pat. No. 6,498,690 B1 discloses a method for imaging assays with an objective comprising a telecentric lens. U.S. Pat. No. 6,246,525 B1 claims an imaging device for imaging a sample carrier comprising a Fresnel lens.
Thus, it was the object of the present invention to provide an improved device for simultaneous monitoring of fluorescence signals from a lateral distribution of sites by optimizing the optical path towards homogeneous illumination and accurate detection. In one aspect of the present invention, the problem to be solved relates to improvements in monitoring multiplexed real-time PCR in a microtiter plate format.