HK1091904B - Imaging fluorescence signals using telecentric optics - Google Patents

Description

Imaging fluorescence signals using telecentric optics

Technical Field

The present invention relates to the field of DNA analysis. In particular, the invention relates to a device for parallel imaging of fluorescence intensity at multiple sites (sites).

Background

Various applications of using fluorescence techniques to analyze biological samples are known to those skilled in the art. In the case of electrophoretic techniques, proteins or DNA are labeled with fluorescent probes to visualize their electrophoretic bands in a colloid or in a column. In addition, most biochip applications to date are based on fluorescent readout, in which the specific binding of fluorescently labeled target molecules to probe molecules immobilized on a solid support is monitored. Applications of DNA analysis in liquid phase include fluorescent hybridization probes like the double-stranded DNA binding dye SybrGreenI or FRET (fluorescence resonance energy transfer) probes using two fluorescent probes and energy transfer. One very important application of fluorescence technology in liquid phase is the real-time quantification of PCR products, also known as real-time PCR.

In all of these cases, a fluorescence reading device is required to provide light of a certain wavelength to excite the fluorescent labels of the assay and to be able to detect the fluorescence forming the labels emitted at a slightly different wavelength. One major problem with all fluorescence reading devices is: the excitation light is of a large intensity compared to the fluorescence emitted by the dye, so in order to accurately monitor the fluorescence signal one must ensure that the excitation beam does not hit the detector. That is, the optical path of the excitation light must be at least partially different from the optical path of the fluorescence light.

The implementation of the fluorescence principle is simple when only one fluorescent probe has to be monitored in the liquid phase, e.g. a capillary. Here, for example, a white light source together with a dichroic mirror and filters is sufficient to meet the requirements. However, if more than one fluorescent label is present in the sample, the lateral distribution of the spots on the solid support or the fluorescence of the microtiter plate has to be monitored, and the requirements of fluorescence reading equipment are therefore more difficult to meet.

In principle, there are two different strategies to excite and monitor the laterally distributed fluorescence at each site. The first strategy is to scan the lateral distribution of sites, thereby analyzing individual sites one at a time in succession. The second strategy is to illuminate the entire distribution of sites simultaneously and image the corresponding fluorescence, for example, on a CCD chip. The scanning strategy has significant drawbacks: either the carrier has to be moved in two dimensions (WO 03/069391, DE 10200499) or the detector has to be moved relative to the carrier (US 2002/159057). On the other hand, the main difficulty with strategies that irradiate the whole carrier simultaneously is to ensure uniform irradiation of the whole distribution of sites. Another alternative to uniform illumination of the entire distribution of sites is to use an array of light sources whereby each site is illuminated by its own light source. DE 10131687 describes this strategy for evaluating PCR in a thermocycler with multiple wells using a beam splitter and an LED array for illumination. DE 10155142 describes dark-field monitoring of fluorescence signals, in which a microarray is also illuminated by an LED array, but in this embodiment no beam splitter is required.

Again, two different possibilities exist, considering the requirement to at least partially separate the optical path of the excitation beam and the optical path of the fluorescence light. The first possibility is called surface illumination (epi-illumination), whereby a beam splitter is utilized and the excitation beam and the fluorescence light share at least part of the optical train. A second possibility is to use oblique illumination. Here, the excitation beam is arranged in such a way that it makes an angle with the normal of the carrier surface and the corresponding reflection of the excitation beam is outside the acceptance angle of the detection system (e.g. US 2002/0005493 a1, EP1275954a 2).

US 2003/0011772 a1 describes an optical device that uses a beam splitter to simultaneously view multiple fluorescent dyes in a probe. DE 19748211 a1 discloses a system for monitoring fluorescence signals generated in wells of a microtiter plate simultaneously using a beam splitter, a field lens and a lens array focusing light into each well. The detection is performed by imaging the light on a photodiode array or a CCD chip. The fluorescence collected in this embodiment of the system is dictated by the amount of dye excited by the cone of light of the focusing lens and therefore depends on the fill level of the well. WO 99/60381 proposes an apparatus for monitoring PCR reactions in multiple vials simultaneously in a temperature cycling block. The optics of the instrument again include a beam splitter, a field lens, a vial lens array that focuses the individual beams into each vial, and a detection mechanism that focuses the emitted light onto, for example, a CCD detector. Due to the necessity of vial lens arrays, the size and lateral density of individual sites is limited. JP 2002014044 describes a fluorescence device for monitoring fluorescence generated in a plurality of wells. The optical components include a beam splitter and a lens system to collectively illuminate the wells with light parallel to a depth direction of the wells. However, the image forming optical system concentrates the light to a detection mechanism. US 6,498,690B 1 discloses a method of imaging an assay using an objective lens comprising a telecentric lens. US 6,246,525B 1 provides an imaging device for imaging a sample carrier, the imaging device comprising a Fresnel (Fresnel) lens.

It is therefore an object of the present invention to provide an improved device for simultaneously monitoring laterally distributed fluorescent signals from sites by optimizing the optical path towards uniform illumination and accurate detection. In one aspect of the invention, the problem to be solved relates to improvements in monitoring multiplexed real-time PCR in microtiter plate format.

Disclosure of Invention

The present invention therefore relates to an optical instrument for imaging the fluorescence of an assembly (assembly) of a plurality of individual sites, comprising excitation across the whole of the assembly area and accurate imaging of the corresponding fluorescence signal.

More precisely, the invention relates to an optical instrument for simultaneous real-time analysis of multiple PCR amplifications occurring in wells of a microtiter plate or imaging the fluorescence intensity of a microarray as a measure of specific target/probe interactions.

One subject of the invention is an optical instrument for imaging fluorescence signals from a plurality of individual sites, comprising:

a holding mechanism 1 for holding an assembled planar carrier 2 with a plurality of individual sites 3,

at least one light source 4 emitting light, comprising at least one excitation frequency,

a transducer 5 arranged to receive the assembled fluorescent signals from the plurality of individual sites 3, wherein the transducer 5 produces raw data that can be calculated,

a field lens 6 that transfers excitation light from the light source 4 to the assembly of the plurality of individual sites 3 and transfers fluorescence signals from the assembly of the plurality of individual sites 3 to the transducer 5,

an excitation lens arrangement 10 which transfers excitation light from the light source 4 to the field lens 6, an

An imaging lens arrangement 11 which transfers the fluorescence signal from the field lens 6 to the transducer 5,

wherein the imaging of the excitation light and fluorescence signals from a plurality of individual sites is telecentric on the object side of the field lens 6.

In the context of the present invention, the assembly of multiple individual sites generalizes an object consisting of two or more spatially separated and laterally distributed sites. The site may be, for example, a well of a microtiter plate or a functionalized surface region of a slide. In most cases the assembly of the multiple individual sites will be arranged in a consistent manner and each site will have different content in order to perform multiplexed analysis. Within the scope of the present invention, said assembled planar support is a planar solid phase. In the case of a microarray, the assembled planar support is the surface of the planar solid phase where the sites are arranged. In the case of a microtiter plate, the assembled planar carrier is a plane where the openings of the wells are arranged. In order to stabilize the position of each individual site at a desired position within the optical path, the assembled planar carrier is held by a holding mechanism.

The phrase Light Source (LS) includes within the scope of the present invention luminaries emitting light of a single frequency or a plurality of different frequencies. Additionally, the light source may be an arrangement of more than one said light emitters.

In the context of the present invention, a transducer (Det) is a device capable of converting visible light into an electrical signal that can be processed by a computer, such as a CCD chip.

Within the scope of the invention, telecentric optics are optics with very small apertures, thus providing high depth of focus. That is, because all points in object and/or image space traversing the object are parallel to the optical axis, the telecentric light of the telecentric optics is quasi-parallel to the chief ray. The quality of the excitation optics or the imaging optics, which exploit the telecentricity in the object space, is therefore insensitive to the distance of a certain object point from the optics. The aperture of the telecentric optics is imaged at infinity. In addition, the use of telecentric light ensures good lateral uniformity across the beam, and the sites at the center of the assembly are comparable to the sites at the boundaries of the assembly. Throughout the present invention, telecentric optics always include a field lens. The field lens, which in the context of the present invention is the single lens closest to the objective lens, determines the field of view of the instrument, comprises one or more components (achromatic lenses) and, in combination with additional optical components of the device, contributes to the telecentricity in the object and/or image space.

The field lens of the present invention transfers excitation light from the light source to the assembly of the plurality of individual sites and transfers assembled fluorescence signals from the plurality of individual sites to the transducer. This does not exclude that additional optical components are introduced into the beam trajectory, for example between the light source and the field lens, between the field lens and the transducer or between the field lens and the assembly of the plurality of individual sites.

Another aspect of the invention is a real-time PCR instrument comprising:

an optical instrument according to the invention, and

a mechanism for heating and cooling a carrier with one or more wells, each well containing a reaction mixture capable of performing a PCR reaction.

Within the scope of the present invention, said means for heating and cooling comprise any means capable of controlling and altering the temperature of the assembly of said plurality of individual sites in a cyclic manner in order to perform a cyclic PCR amplification of nucleic acids. Preferably, the holding mechanism may be heated and cooled in thermal contact with the assembled planar carrier of the plurality of individual sites.

Yet another aspect of the invention is a system for imaging fluorescence signals of a plurality of analytes, comprising:

a planar carrier 2 comprising an assembly of a plurality of individual assays,

at least one light source 4 emitting light, comprising at least one excitation frequency,

a transducer 5 arranged to receive fluorescent signals from the plurality of analytes, wherein the transducer produces raw data that can be calculated, an

A beam path from the light source 4 to the transducer 5, characterized by telecentric excitation of the assembly of the plurality of individual analytes and telecentric imaging of the fluorescence signal produced at each individual analyte of the assembly of the plurality of individual analytes.

The assembly of multiple individual assays summarizes an object composed of two or more assays that are spatially separated to enable parallel analysis. These assays may be performed, for example, in wells of a microtiter plate or on functionalized surface areas of a slide.

The phrase beam trajectory is used throughout the present invention to summarize all regions traversed by a light beam from the light source at least through the field lens to the assembly of the plurality of individual assays and from the assembly of the plurality of individual assays at least through the field lens en route to the transducer.

Another subject of the invention is a system for simultaneously performing and monitoring a plurality of PCR reactions in real time, comprising:

a multi-well plate with a plurality of individual sites, each site containing a reaction mixture capable of performing a PCR reaction,

a fluorescent DNA conjugate, and

a real-time PCR instrument according to the invention comprises an optical instrument according to the invention which illuminates the entire multi-well plate with telecentric light and detects fluorescence signals from each well of the multi-well plate by means of a transducer which is arranged to receive the corresponding fluorescence signals in order to generate calculable raw data.

Throughout the present invention, the fluorescent DNA binding entity is a fluorescent dye or an assembly of fluorescent dyes known to some skilled in the art, which can be used for the detection of amplified DNA, i.e. for example a double stranded DNA binding dye, a TagMan probe, a molecular beacon, a single labelled probe or a FRET hybridisation probe.

A further subject of the present invention is a method for amplifying, detecting and/or quantifying a plurality of target DNA sequences, comprising:

there is provided a composition or reaction mixture capable of performing a PCR reaction,

subjecting the reaction mixture to a thermal cycling protocol such that amplification of the plurality of target DNA sequences can occur, an

The presence and quantity of each DNA sequence is monitored at least once after the course of a plurality of amplification cycles using a fluorescent DNA binder and a real-time PCR instrument according to the invention.

The composition or reaction mixture capable of performing a PCR reaction includes, throughout the present invention, buffers, nucleotides, enzymes, primers, and fluorescent DNA conjugates.

A thermal cycling protocol is a protocol that defines the chronological temperature treatment, melting and annealing temperatures, number of amplification cycles, and time of heating and cooling of the PCR composition.

Drawings

FIG. 1 is a schematic view of one embodiment of an optical instrument according to the present invention.

Fig. 2 is a schematic view of another embodiment of an optical instrument according to the present invention.

Detailed Description

One aspect of the invention is an optical instrument for imaging fluorescence signals from a plurality of individual sites, comprising:

a holding mechanism 1 for holding an assembled planar carrier 2 with a plurality of individual sites 3,

at least one light source 4 emitting light, comprising at least one excitation frequency,

a transducer 5 arranged to receive the assembled fluorescent signals from the plurality of individual sites 3, wherein the transducer 5 produces raw data that can be calculated,

a field lens 6 that transfers excitation light from the light source 4 to the assembly of the plurality of individual sites 3 and transfers fluorescence signals from the assembly of the plurality of individual sites 3 to the transducer 5,

an excitation lens arrangement 10 which transfers excitation light from the light source 4 to the field lens 6, an

An imaging lens arrangement 11 which transfers the fluorescence signal from the field lens 6 to the transducer 5.

Wherein the imaging of the excitation light and fluorescence signals from a plurality of individual sites is telecentric on the object side of the field lens 6.

Those skilled in the art know that a large number of instruments can be used to image the fluorescence signal. If the optical instrument should be able to image the assembled fluorescent signal of multiple individual sites simultaneously, e.g. wells of a microtiter plate or spots of a microarray, one has to ensure that the excitation of the dye and the imaging of the fluorescent signal at the center of the assembly and at the borders of the assembly are comparable. Furthermore, even if the requirement of uniformity of the intensity distribution across the beam is met, alignment of the planar carrier is important in order to ensure that the carrier as a whole is in the focal plane of the imaging optics and the excitation optics. When the carrier has a depth, similar to the case of a microtiter plate, some special problems arise in addition.

One solution to the above problem is to use telecentric optics. In a telecentric optic, the focal plane is at infinity and the chief rays emanating from each object point are parallel to the optical axis. Thus, all object points within a limited field of view are observed to have the same perspective and the same intensity, in other words, the telecentric optics has a large depth of field and a uniform excitation or imaging profile.

Telecentric optics can be characterized by its Numerical Aperture (NA), which should be as small as possible to achieve a high depth of focus:

NA＝n sin A，

where n is the refractive index of the medium and A is the aperture angle. If the assembly of individual sites has a certain depth, as is the case with microtiter plates, a high depth of focus is of utmost importance.

In order to design an optical instrument for telecentric excitation of the lateral distribution of sites and telecentric imaging of the fluorescence signals from the sites, one must consider several aspects. The NA value should be as small as possible, solely in terms of depth of focus. On the other hand, a small NA value for the imaging optics corresponds to a poor imaging resolution, while a small NA value for the excitation optics corresponds to a waste of illumination power for the excitation.

If the telecentric optics should be usable over the entire frequency range, the optics must also be achromatic. For fluorescence imaging itself, even more requirements have to be dealt with, since fluorescence imaging has to have a suitable scaling to correctly reproduce the lateral distribution of sites on the transducer. In addition, aberrations like spherical or chromatic aberration, coma, astigmatism or curvature of field have to be controlled.

There are several ways to create telecentric optics. Typically, telecentric optics are multi-element lens designs in which more than one lens is arranged in series in the beam path. Telecentric optics can be prepared to be telecentric in the object plane or telecentric in the image plane, or both, a so-called double telecentric optics. Furthermore, it is possible to illuminate the object with telecentric light and/or to monitor the object in a telecentric manner. It is generally sufficient to provide an optical device having telecentricity in the object plane, since this already ensures a uniform illumination of the entire object in the lateral direction as well as in the third dimension and an accurate collection of the light radiated from the object.

From the state of the art, instruments are known which use telecentric optics for imaging the fluorescence signal, but the excitation is usually performed in a non-telecentric manner, for example by back-illumination, oblique illumination or by evanescent fields. Throughout the present invention, both the excitation of the plurality of individual sites and the imaging of the fluorescence signals from the plurality of individual sites are performed in a telecentric manner.

Fig. 1 and 2 show schematic views of two optical instruments according to a preferred embodiment of the present invention, which will be described in detail below.

The central portion of all telecentric optics is the field lens. The lens is closest to the object and determines the diameter of the instrument's field of view. Therefore, when the assembly of multiple individual sites is distributed over a large area, the diameter of the lens tends to increase in size. The field lens is in the form of a single piece (a single lens) or an achromatic lens, e.g. comprising two lenses glued together. A particular field lens that can be used in the present invention is a fresnel lens. The fresnel lens has a particular complex curvature with a plurality of tapered regions on at least one optically active surface that provide the same telecentric properties as the field lens. In most cases, a fresnel lens has only one surface with a plurality of tapered zones, which is supported by a planar surface perpendicular to the optical axis, so that they are thinner than normal field lenses. In special cases, a fresnel lens is provided additionally with a curved bearing surface or with a plurality of conical regions on both sides of the lens. In addition, fresnel lenses are sometimes made of plastic, so they are less expensive than large field lenses made of glass. On the other hand, however, the image quality of these fresnel lenses is lower than that of normal field lenses, especially when contrast and crosstalk are taken into account, because light scattering occurs at those points of the lenses with discontinuous curvature.

In a preferred embodiment, the optical instrument according to the invention further comprises a beam splitter 7 transparent for at least one excitation frequency and reflective for the frequency of the fluorescence signal, or a beam splitter reflective for at least one excitation frequency and transparent for the frequency of the fluorescence signal.

The beam splitter is typically a dichroic mirror that passes or reflects light depending on its wavelength, so it can be used to spatially separate two components of a light beam into different directions. Such dichroic mirrors may be made of glass or plastic if necessary with some optically active coating. They are present in the form of thin foils or prisms.

For application in optical instruments to image fluorescent signals, the dichroic mirror must be reflective for the excitation light and transparent for the fluorescence (fig. 2) or vice versa (fig. 1). The separation of the light emitted from the light source into a beam comprising one or more excitation frequencies and a beam with its frequency helps to ensure that the fluorescent dye is not destroyed by short wavelengths and that undesired background radiation, for example generated by excitation of the carrier, is reduced. The separation of light from the plurality of individual sites into components comprising at least one excitation frequency and components comprising a fluorescent signal avoids reflections of excitation light with high intensity from hitting the transducer. This greatly improves the signal-to-noise ratio.

In another preferred variant of the invention, the field lens generates an excitation beam perpendicular to the assembled carriers of the plurality of individual sites.

The assembled support with the excitation beam perpendicular to the plurality of individual sites also produces a reflected beam perpendicular to the assembled support. But due to the beam splitter, the reflected beam is separated from the fluorescence signal and does not hit the transducer. In the case of, for example, a multi-titer plate as an assembly of the plurality of individual sites, the vertical excitation beam has the advantage that: it is able to penetrate the depth of the well. On the other hand, if the excitation beam reaches the carrier at an angle of incidence greater than 0 °, the walls of the well will hide the full illumination inside the well and only a part of the fluorochromes can be excited. Furthermore, when using an oblique excitation beam, the amount of fluorochrome excitation within the well depends on the fill level.

In a further preferred variant, the optical instrument according to the invention further comprises an excitation filter system 8 capable of shifting at least one excitation frequency from said light source to the assembly of said plurality of individual sites, while blocking a plurality of other frequencies.

Such an additional excitation filter system may even block certain frequencies from the light source before the beam splitter. This is necessary if the light source comprises light whose frequency cannot be separated from the excitation frequency by the beam splitter. One suitable excitation filter system is for example referred to as a filter wheel, which comprises a number of individual filters with different optical properties. The use of such a filter wheel provides a simple means to vary the excitation frequency. Special excitation filter systems are for example filters that absorb Infrared (IR) frequencies or Ultraviolet (UV) light. Such special excitation filter systems may be implemented in the form of separate optical components such as thin film filters or in the form of optically active coatings on other optical components of the device.

In a further preferred embodiment, the optical instrument according to the invention further comprises an imaging filter system 9 capable of transferring the assembled fluorescence signals from the plurality of individual sites to the transducer while blocking light having an excitation frequency.

Such additional imaging filtering systems may also block certain frequencies generated at the plurality of individual sites or from the excitation reflections after the beam splitter. This is necessary if light is generated at the plurality of individual sites whose frequencies cannot be separated from the excitation frequency by the beam splitter. Again, one suitable imaging filtering system is a filter wheel containing different filters. Similar to the case of excitation filter systems, the special imaging filter system may be, for example, an Infrared (IR) filter or an Ultraviolet (UV) filter. The special imaging filter system may be implemented in the form of a separate optical component, such as a thin film filter, or in the form of an optically active coating on other optical components of the device. Another imaging filter system is a filter system that avoids the detection of scattered light by the detector.

As mentioned above, the optical instrument according to the invention comprises an excitation lens arrangement 10 which diverts light from the light source 4 to the field lens 6.

This means that the light from the light source is imaged on the assembly of the plurality of individual sites using the excitation optics comprising the field lens 6 and the excitation lens arrangement 10. The excitation optics provide telecentric excitation light on the object side of the field lens 6 and are therefore telecentric excitation optics. The excitation lens arrangement includes at least one lens, preferably at least three lenses, to increase the excitation aperture towards better utilization of the light source power. The excitation lens arrangement may comprise an aspherical surface if the number of lenses should be reduced. Preferably, the telecentric excitation optics is designed to be achromatic in order to achieve an assembled uniform intensity distribution across the plurality of individual sites independent of the excitation wavelength.

In another embodiment of the invention, the light source emits light comprising a plurality of frequencies, preferably the light source is a white light source, most preferably the light source is a gas discharge lamp, for example a xenon or mercury lamp, or an incandescent lamp, such as a tungsten filament lamp.

In yet another embodiment of the invention said light source emits light with a single frequency, preferably said light source is a laser, most preferably said light source is an LED.

The use of light sources emitting light with different frequencies has the advantage that: the light source can be used for different fluorescent dyes simply by changing the filter set, which consists of the beam splitter and, if necessary, also the excitation filter system and/or the imaging filter system. In order to easily switch from one fluorescent dye to another, it is preferred to use a filter wheel as the excitation filter system and/or the imaging filter system that contains a certain amount of filters. On the other hand, if the light source emits light with only a single frequency, the requirements of this filter set are easily met, but the optical instrument is fixed to a limited amount of fluorescent dye.

In one embodiment of the invention, the light source window has an optically active coating that acts as a special excitation filter system to absorb IR and/or UV light.

In a further preferred variant of the invention, the light source comprises a combination of more than one light emitter, preferably a combination of more than one laser, most preferably a combination of more than one LED.

In this preferred embodiment, in order to provide an optical instrument according to the invention with more than one excitation frequency, an assembly of different luminophores is used. An embodiment according to the invention with two different light sources, each with its own excitation filter system 8, excitation lens arrangement 10 and beam splitter 7, is shown in fig. 2.

In another variant according to the invention said light source further comprises a device for selecting one or more of said luminaires.

The device for selecting one of the luminaires can be implemented in different ways. One possibility is to use a rotatable mirror to inject the light of the selected luminous body into the optical path. Another possibility is to move the arrangement of the luminaires in order to inject the light of the selected luminaire into the optical path.

In addition to the field lens 6, the excitation filter system 8, the excitation lens arrangement 10 and the beam splitter 7, the telecentric excitation optics according to the invention may also comprise several additional components. In one embodiment the telecentric excitation optics additionally comprises a light guide, and light from the light source is coupled to the light guide for transferring the light from the light source to the optical components of the optical system. Using a light guide it is possible to couple light from different light sources and simultaneously transfer the combined light to the optical component. All types of light guides are applicable for the purposes of the present invention. Possible light guides are, for example, fluid light guides, fiber light guides or fiber light guide bundles. In one embodiment of the invention, one or both ends of the light guide have an optically active coating that acts as a special excitation filter system to absorb IR and/or UV light.

In yet another embodiment according to the present invention, the telecentric excitation optics further comprises a light mixer to mix light from the light source and image an illuminated surface of the light mixer onto the assembly of the plurality of individual sites.

A light mixer is a device with a very uniformly illuminated surface that can be used as a light source to provide light with a uniform intensity distribution over the whole cross-section. The light mixer is a slightly elongated solid body made of an optically transparent material, wherein the boundaries of said solid body are parallel to said light path. That is, the light mixer is an optical fiber. The light injected into the light mixer undergoes multiple total reflections on the inner surface of the optically transparent material, which creates a very uniformly illuminated cross-sectional area at one end of the optical fiber. The total reflection at the inner interface of the optically transparent material is simply based on a change in refractive index at the interface or may be supported by a refractive coating. The ratio of the length of the light mixer to its cross-sectional area is important for illumination uniformity. Said ratio is preferably greater than 2.

Light from the light source, in particular light from the cross-sectional area of one end of the light mixer, is imaged onto the assembly of the plurality of individual sites by using telecentric excitation optics comprising a field lens 6 and an excitation lens arrangement 10. Therefore, in this embodiment of the invention, the excitation of the plurality of individual sites is performed with excitation optics that are telecentric at the object site of the field lens 6.

The optical device according to the invention is also suitable for imaging chemiluminescence and bioluminescence. Since excitation light is not required in these cases, the light source 4, the excitation lens arrangement 10 and the excitation filter system 8 may be omitted.

In a further preferred variant, the optical instrument according to the invention further comprises a beam folding unit comprising one, two or more folding mirrors, said folding unit folding the light from the light source and the assembled fluorescent signals from the plurality of individual sites.

A beam folding unit is a unit within the scope of the invention that provides a long optical path while requiring only a limited amount of space. One parameter that can be modified in order to adjust the numerical aperture from the excitation optics is the optical path that the light must travel. Enlarging the optical path reduces the numerical aperture. So, if a small aperture is desired to meet the depth of field requirement and uniform intensity distribution, the optical path will be longer. Due to the unconditional inadequacy of large instruments, the folding mirror can be used to achieve a long optical path and at the same time limit the instrument size.

As mentioned before, the optical instrument according to the invention comprises an imaging lens arrangement 11, said imaging lens arrangement 11 transferring light from said field lens 6 to said transducer 5.

This means that the fluorescence signal generated at the assembly of the plurality of individual sites is imaged on the transducer 5 by telecentric imaging optics comprising the field lens 6 and the imaging lens arrangement 11. In other embodiments of the invention, the telecentric imaging optics further comprise, for example, a beam folding unit and/or a special imaging filter system 9.

The telecentric imaging optics must be optimized in terms of the size of the transducer and the assembled spatial dimensions of the plurality of individual sites. As in the case of the excitation lens arrangement 11, the imaging lens arrangement 11 comprises an assembly of at least one lens, preferably at least 5 lenses. The imaging lens arrangement requires a large number of lenses, since the imaging optics have to cope with even higher requirements than the excitation optics. The fluorescence imaging must have the proper scaling to correctly reproduce the lateral distribution of sites on the transducer. In addition, aberrations like spherical or chromatic aberration, coma, astigmatism, special errors or curvature of the image field have to be controlled. Since the fluorescence signal is imaged onto the transducer, the fluorescence imaging is performed with an imaging optics which is telecentric only at the object location of the field lens 6.

In a further preferred variant of the optical instrument according to the invention, the imaging lens arrangement 11 is coupled to the transducer 5 to form an imaging unit 12.

Note that in this preferred embodiment of the invention, the telecentric imaging optics is different from a standard objective lens, in which all lenses are arranged and fixed in a defined manner to form an objective lens and the objective lens as a whole is placed between the transducer and the object. In this preferred embodiment of the invention, on the contrary, the imaging lens arrangement 11 is coupled to the transducer 5, thereby forming an imaging unit 12. The positioning of the imaging lens arrangement and the transducer is particularly important in order to meet requirements with regard to imaging resolution and accuracy. In this embodiment, these requirements are met by optimizing the position between the imaging lens arrangement and the transducer before the optimized position is fixed. The coupling between the imaging lens arrangement and the transducer is maintained throughout the intended use and is only released when a re-optimization of the positioning becomes necessary.

In an embodiment of the optical instrument according to the invention, the transducer comprises a semiconductor device or preferably a charge coupled device.

In the context of the present invention, a transducer is a device capable of converting light into an electrical signal that can be processed by a computer. This may be achieved by a semiconductor device having a bandgap that is less than the energy corresponding to the fluorescent signal to be detected. Electrons generated in the conduction band of the semiconductor by irradiation of the device produce a measurable signal that can be converted into calculable data. Examples of such semiconductor devices are photodiodes or Charge Coupled Devices (CCDs).

A further preferred variant of the optical device according to the invention is an optical device wherein the assembled individual sites are wells, the excitation light is parallel to the side walls of the wells and the solution filling the wells comprises a fluorescent dye.

An example of a further preferred variant of the optical instrument is an apparatus for simultaneously monitoring PCR (polymerase chain reaction) amplifications taking place in individual wells of a microtiter plate. In order to illuminate the entire interior of the well independently of the fill level within the well, the excitation light is parallel to the side walls of the well. Since telecentric optics for excitation as well as for fluorescence imaging are used, the results obtained from wells in the center of the plate are comparable to those obtained from wells at the borders of the plate.

In case PCR amplification is performed in a single well, all fluorophores can be applied as fluorescent dyes that specifically bind to double stranded nucleic acids. In the context of the present invention, these fluorescent dyes are referred to as fluorescent DNA binders, wherein the fluorescent DNA binders are molecules or a pair of molecules providing a characteristic fluorescence if they bind to double stranded DNA. The following detection formats are well known in the field of real-time PCR monitoring: DNA binding dye formats (e.g., SybrGreenI), TaqMan probes, molecular beacons, single-label probe (SLP) formats, or FRET hybridization probes.

A further preferred embodiment of the optical device according to the invention is an optical device, wherein said assembled individual sites are spots on a planar carrier and a fluorescent dye is attached to said spots.

An example of this preferred embodiment of the optical instrument is a device for simultaneously imaging fluorescence signals from different spots of a planar array. In a particular embodiment such an array is a DNA array, wherein the lateral restriction regions are functionalized with DNA probes having different sequences. In this case, the optical instrument according to the invention can monitor hybridization events with a sample comprising nucleic acids if, for example, the complementary DNA strands are labeled with a fluorescent dye. As an alternative to the labeling of DNA molecules in the sample, hybridization events can also be visualized by double-stranded nucleic acids in combination with fluorescent dyes.

The present invention also relates to a real-time PCR instrument comprising:

an optical instrument according to the invention, and

a mechanism to heat and cool a carrier having one or more wells, each well containing a reaction mixture capable of performing a PCR reaction.

Within the scope of the present invention, said means for heating and cooling comprise any means capable of controlling and varying the temperature of the assembly of said plurality of individual sites in a cyclic manner in order to perform a cyclic PCR amplification of nucleic acids. Each PCR cycle includes several different steps: a reduced temperature annealing step, an enzymatic amplification step at a relatively low temperature in conjunction with a detection step using a fluorescent dye and a melting step at a high temperature.

The invention further relates to a system for imaging fluorescence signals of a plurality of analytes, comprising:

a planar carrier 2 comprising an assembly of a plurality of individual assays,

at least one light source 4 emitting light, comprising at least one excitation frequency,

a transducer 5 arranged to receive fluorescent signals from the plurality of analytes, wherein the transducer produces raw data that can be calculated, an

A beam path from the light source 4 to the transducer 5, characterized by telecentric excitation of the assembly of the plurality of individual analytes and telecentric imaging of the fluorescence signal produced at each individual analyte of the assembly of the plurality of individual analytes.

The assembly of multiple individual assays summarizes an object composed of two or more assays that are spatially separated to enable parallel analysis. These assays may be performed, for example, in wells of a microtiter plate or on functionalized surface areas of a slide. In most cases the assembly of the plurality of individual assays will be arranged in a consistent manner and each assay will have different content in order to perform multiplexed analysis. In the case of DNA microarrays, each spot of the array is functionalized with an oligomer having a certain sequence, wherein in the case of immunoassays each spot of the array is functionalized, for example, with proteins having different affinities. In the case of microtiter plates, for example, different PCRs are carried out in each well.

In a preferred embodiment of the system for imaging fluorescence signals of a plurality of analytes according to the invention, the system further comprises a field lens, wherein the beam trajectory passes the field lens twice.

In another preferred embodiment of the system for imaging fluorescence signals of a plurality of analytes according to the invention, the system further comprises an imaging lens arrangement 11, wherein the imaging lens arrangement 11 is coupled to the transducer 5 to form an imaging unit 12.

In yet another preferred embodiment of the system for imaging fluorescence signals of a plurality of analytes according to the invention, the system further comprises a beam splitter 7 being transparent for at least one excitation frequency and being reflective for the frequency of the fluorescence signal, or comprises a beam splitter 7 being reflective for at least one excitation frequency and being transparent for the frequency of the fluorescence signal.

Yet another preferred embodiment of a system for imaging fluorescence signals of a plurality of analytes according to the invention further comprises an excitation filter system capable of transferring at least one excitation frequency from the light source to the assembly of the plurality of individual sites and simultaneously blocking a plurality of other frequencies and/or an imaging filter system capable of transferring assembled fluorescence signals from the plurality of individual sites to the transducer and simultaneously blocking light having an excitation frequency.

Another aspect of the invention relates to a system for simultaneously performing and monitoring multiple PCR reactions in real-time, comprising:

a multi-well plate with a plurality of individual sites, each site containing a reaction mixture capable of performing a PCR reaction,

a fluorescent DNA conjugate, and

a real-time PCR instrument comprising an optical instrument according to the invention which illuminates the entire multi-well plate with telecentric light and detects fluorescence signals from each well of the multi-well plate by a transducer arranged to receive the respective fluorescence signals so as to produce raw data which can be calculated.

Generally, there are two forms of fluorescent DNA conjugates for real-time detection of amplified DNA, of which the following are well known and commonly used in the art:

a) DNA binding dye forms

Since the amount of double-stranded amplification product usually exceeds the amount of nucleic acid originally present in the sample to be analyzed, double-stranded DNA-specific dyes can be used which exhibit enhanced fluorescence upon excitation with a suitable wavelength only when they bind to double-stranded DNA. Preferably, only those dyes like SybrGreen I that do not affect the efficiency of the PCR reaction can be used.

All other means known in the art require the design of a fluorescently labeled hybridization probe that fluoresces only after binding to its target nucleic acid.

b) TaqMan probe

Single-stranded hybridization probes are labeled with two components. When the first component is excited with light of the appropriate wavelength, the absorbed energy is transferred to a second component called the quencher, according to the principle of fluorescence resonance energy transfer. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5 '-3' exonuclease activity of Taq polymerase for a subsequent extended period. As a result, the excited fluorescent component and the quencher are spatially separated from each other, and thus the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,538,848).

c) Molecular beacons

These hybridization probes are also labeled with a first component and a quencher, the labels preferably being located at both ends of the probes. As a result of the secondary structure of the probe, the two components are in spatial proximity in solution. The two components are separated from each other after hybridization to the target nucleic acid, so that after excitation with light of a suitable wavelength, the fluorescence emission of the first component can be measured (U.S. Pat. No. 5,118,801).

d) Single Label Probe (SLP) Format

The detection format consists of a single oligonucleotide labeled with a single fluorescent dye at the 5 '-or 3' -end (WO 02/14555). Two different designs can be used for oligo-labelling: g-quenching probe and nitroindole quenching probe.

In the G-quench probe example, the fluorescent dye is attached to the C at the 5 '-or 3' -end of the oligo. If two G's are located on the target strand opposite C and at position 1 flanking the complementary oligonucleotide probe, fluorescence is significantly reduced when the probe is hybridized to the target.

In the nitroindole quench probe example, the fluorescent dye is attached to the nitroindole at the 5 '-or 3' -end of the oligonucleotide. Nitroindole reduced the fluorescent signaling of the free probe to some extent. Fluorescence increases due to quenching when the probe is hybridized to the target DNA.

e) FRET hybridization probes

The FRET hybridization probe test format is particularly useful for all types of homogeneous hybridization assays (Matthews, J.A., and Kricka, L.J., anal. biochem., analytical biochemistry 169(1988) pages 1-25). Characterized by a pair of two single-stranded hybridization probes that are used simultaneously and are complementary to adjacent sites on the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When excited with light of a suitable wavelength, the first component transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer, so that when two hybridization probes bind to adjacent locations of the target molecules to be detected, the fluorescence emission of the second component can be measured.

When annealing to a target sequence, the hybridization probes must be positioned in close proximity to each other in a head-to-tail arrangement. Typically, the gap between the labeled 3 '-end of the first probe and the labeled 5' -end of the second probe is as small as possible, i.e., 1-5 bases. This allows the FRET donor compound and the FRET acceptor compound to be in close proximity, which is typically 10-100 angstroms.

Instead of monitoring the increase in fluorescence of the FRET acceptor component, it is also possible to monitor the decrease in fluorescence of the FRET donor component as a quantitative measure of the hybridization event.

In particular, for detection of amplified target DNA, the FRET hybridization probe format can be used for real-time PCR. Among all the detection formats known in the field of real-time PCR, the FRET hybridization probe format has proven to be highly sensitive, accurate and reliable (WO 97/46707; WO 97/46712; WO 97/46714). However, the design of suitable FRET hybridization probe sequences can sometimes be limited by the particular characteristics of the target nucleic acid sequence to be detected.

As an alternative to the use of two FRET hybridization probes, it is also possible to use fluorescently labeled primers and only one labeled oligonucleotide probe (Bernard, P.S. et al, anal. biochem. "analytical biochemistry" 255(1998) page 101-107). In this regard, it may be arbitrarily selected regardless of whether the primer is labeled with a FRET donor or FRET acceptor compound.

The present invention further relates to a method for amplifying, detecting and/or quantifying a plurality of target DNA sequences, comprising:

providing a composition or reaction mixture capable of performing a PCR reaction,

subjecting the reaction mixture to a thermal cycling protocol such that amplification of the plurality of target DNA sequences can occur, an

The presence and quantity of each DNA sequence is monitored at least once after a number of amplification cycles using a fluorescent DNA binder and a real-time PCR instrument according to the invention.

Examples, references and figures are provided below to assist in understanding the invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the procedures set forth without departing from the spirit of the invention.

Examples of the present invention

An optical instrument as described in the detailed description and shown in fig. 2 (with only one light source) is configured as follows. The telecentric excitation optics is tuned to handle frequencies of 450-650nm and the telecentric imaging optics handles frequencies of 500-740 nm. The light source was a xenon lamp and as a transducer a cooled 2/3 "CCD chip with 1024 x 1344 pixels was used. The optical instrument is designed to image an area of 83mm x 117mm, so that a microtiter plate (MTP) with 96 wells (distance 9 mm; diameter 5mm) and 384 wells (distance 4.5 mm; diameter 3mm) can be used. For certain fluorescent dyes, the appropriate wavelengths for excitation and imaging are adjusted by a filter wheel.

The telecentric excitation optics has a numerical aperture of 0.35 on the side of the light source and a numerical aperture of 0.014 on the side of the MTP. The light source is arranged perpendicular to the CCD chip and the excitation beam must be directed towards the MTP by a beam splitter that is reflective for the necessary excitation frequencies and transparent for other frequencies contained in the light from the light source. The excitation beam from the beam splitter is perpendicular to the MTP and has an intensity variation of 10% or less across the object field (88mm x 122 mm). The imaging optics also has an aperture of 0.014 on the object side and a regeneration ratio of-0.075 for an 800mm object distance. This large distance is achieved using two folding mirrors. The imaging optics have a depth of field of +/-3 mm. Due to the excitation, the beam splitter used is transparent to the fluorescence signal generated in the MTP well.

List of references

Analytical biochemistry 255(1998) page 101-107 of analytical biochem, Bernard, P.S., et al

DE 10131687

DE 10155142

DE 10200499

DE 19748211 A1

EP 1275954 A2

JP 2002014044

Anal. biochem, Matthews, J.A. and Kricka, L.J. [ analytical biochemistry ] (1988) pp.1-25

US 2002/0005493 A1

US 2002/159057

US 2003/0011772 A1

US 5,118,801

US 5,538,848

US 6,246,525 B1

US 6,498,690 B1

WO 02/14555

WO 03/069391

WO 97/46707

WO 97/46712

WO 97/46714

WO 99/60381

Claims (11)

1. An optical instrument for imaging fluorescence signals from a plurality of individual sites, comprising:

a holding mechanism (1) for holding an assembled planar carrier (2) with a plurality of individual sites (3);

at least one light source (4) emitting light, comprising at least one excitation frequency;

a transducer (5) arranged to receive the assembled fluorescent signals from the plurality of individual sites (3), wherein the transducer (5) produces raw data that can be calculated;

a field lens (6) being a single lens and being arranged closest to the plurality of individual sites (3) and transferring excitation light from the light source (4) to the assembly of the plurality of individual sites (3) and transferring assembled fluorescence signals from the plurality of individual sites (3) to the transducer (5);

an excitation lens arrangement (10) that transfers excitation light from the light source (4) to the field lens (6);

an imaging lens arrangement (11) which transfers the fluorescence signal from the field lens (6) to the transducer (5),

wherein the imaging of the excitation light and fluorescence signals from a plurality of individual sites is telecentric on the object side of the field lens (6).

2. The optical instrument according to claim 1, further comprising a beam splitter (7) transparent to at least one excitation frequency and reflective to the frequency of the fluorescence signal, or comprising a beam splitter reflective to at least one excitation frequency and transparent to the frequency of the fluorescence signal.

3. The optical instrument according to one of claims 1 to 2, wherein the imaging lens arrangement (11) is coupled to the transducer (5) to form an imaging unit (12).

4. The optical instrument of one of claims 1-2, wherein the individual site of the assembly is a well, the excitation light is parallel to a sidewall of the well, and the solution filling the well comprises a fluorescent dye.

5. The optical device of one of claims 1-2, wherein the individual sites of the assembly are spots on a planar carrier and a fluorescent dye is attached to the spots.

6. A real-time PCR instrument, comprising:

the optical device according to any one of claims 1-4;

a mechanism for heating and cooling a planar carrier with one or more wells, each well containing a reaction mixture capable of performing a PCR reaction.

7. A system for imaging fluorescent signals of a plurality of analytes, comprising:

a planar carrier (2) comprising an assembly of a plurality of individual assays;

at least one light source (4) emitting light, comprising at least one excitation frequency;

a transducer (5) arranged to receive fluorescent signals from the plurality of analytes, wherein the transducer produces raw data that can be calculated;

characterized in that the system further comprises a field lens being a single lens and being arranged proximate to the plurality of individual analytes, wherein a beam trajectory from the light source (4) to the transducer (5) passes twice through the field lens and the telecentric excitation of the assembly of the plurality of individual analytes and the telecentric imaging of the fluorescence signal generated at each individual analyte of the assembly of the plurality of individual analytes.

8. The system according to claim 7, further comprising an imaging lens arrangement (11), wherein the imaging lens arrangement (11) is coupled to the transducer (5) to form an imaging unit (12).

9. The system according to one of claims 7-8, further comprising a beam splitter (7) being transparent for at least one excitation frequency and being reflective for the frequency of the fluorescence signal, or comprising a beam splitter (7) being reflective for at least one excitation frequency and being transparent for the frequency of the fluorescence signal.

10. A system for simultaneously performing and monitoring multiple PCR reactions in real-time, comprising:

a multi-well plate with a plurality of individual sites, each site comprising a reaction mixture capable of performing a PCR reaction;

a fluorescent DNA conjugate;

a real-time PCR instrument according to claim 6 including an optical instrument according to any of claims 1 to 4 which illuminates the entire multiwell plate with telecentric light and detects fluorescence signals from each well of the multiwell plate by a transducer arranged to receive the respective fluorescence signals so as to produce raw data which can be calculated.

11. A method for amplifying, detecting and/or quantifying a plurality of target DNA sequences, comprising:

providing a composition or reaction mixture capable of performing a PCR reaction;

subjecting the composition or reaction mixture to a thermal cycling protocol such that amplification of the plurality of target DNA sequences can occur;

monitoring the presence and quantity of each DNA sequence at least once after a plurality of amplification cycles using a fluorescent DNA binder and a real-time PCR instrument according to claim 6.