WO2009098624A1

WO2009098624A1 - Analysis system and method

Info

Publication number: WO2009098624A1
Application number: PCT/IB2009/050397
Authority: WO
Inventors: Erik M. H. P. Van Dijk; Marinus I. Boamfa; Reinhold Wimberger-Friedl
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2008-02-04
Filing date: 2009-02-02
Publication date: 2009-08-13

Abstract

An analysis system (101, 102) detects multiple radiation outputs from a sample (400) being analyzed. A radiation source (101, 102) generates input radiation comprising frequency components in multiple frequency bands, with the frequency components being independently controllable. A plurality of detectors (701, 702) are used and a (fixed) filter arrangement (601, 602) with multiple pass bands is associated with each detector. The detector signals can be processed to determine the levels of output radiation from the sample at a plurality of frequencies greater in number than the number of detectors, without having to move any filters.

Description

Analysis system and method

FIELD OF THE INVENTION

The invention relates to an analysis system and an analysis method for analyzing a sample. The invention further relates to a computer programmed product for carrying out the analysis method. The invention may be applied for sample analysis in the fields of chemistry or biochemistry as well in medical analysis or diagnostics using for example luminescence detection.

BACKGROUND OF THE INVENTION An example of the use of fluorescence detection is in nucleic acid testing

(NAT). This is a core element in molecular diagnostics for detecting genetic predispositions for diseases, for determining RNA expression levels or identification of pathogens, like bacteria and viruses that cause infections.

In many cases, particularly in the identification of pathogens, the amount of target DNA present in a reasonable sample volume is very low, and this does not allow direct detection. Amplification techniques are necessary to obtain detectable quantities of the target material. Different amplification techniques have been proposed and are used in daily practice. The most widely used are based on the so-called Polymerase Chain Reaction (PCR). Without going into detail about the different amplification techniques available, a number of these rely on the generation and/or detection of fluorescence of a label substance such as for example a fluorophore in order to detect the presence of the target DNA sequence and/or the progress of amplification. Moreover, in many cases it is desired to conduct multiplexed detection of multiple target DNA sequences for one sample. In a so- called "multiplex PCR" amplification, different target DNA sequences are thus detected. This can be implemented using various fluorescent label substances that have different absorption and emission spectra allowing their distinct optical detection.

To enable such multiplexed detection most known detection systems use a broad spectrum lamp and a single detector and a filter wheel can be used to define different excitation wavelengths and detection windows in order that the different spectral components generated by the label substances can be separately detected in a sequential way. There are systems that use up to four different excitation wavelengths from one lamp, and different detectors to be able to detect the different labels.

However, the known system becomes bulky, and for every increase in the degree of multiplexing an extra filter has to be added.

A known system named the "StepOne system" has been proposed by Applied Biosystems. It uses a single blue LED and three filters to sequentially detect up to three different labels. In this case, the single diode excites all the different fluorophores (FAM, JOE, ROX) simultaneously, but their emission spectra are shifted within the wavelength range with respect to each other so that separated detection is allowed. This in principle provides a more compact solution since only a single filter in front of the detector is rotated or switched.

However, one major problem of the "StepOne system" is the fact that in practice there is significant cross talk between the spectra of the different fluorophores. The absorption and emission spectra are shown in Fig. 2a and 2b respectively. For any detection window selected (corresponding to the peaks in Fig. 2b), there will be considerable cross talk between the contributions of the different fluorophores. By careful calibration, in principle the relative contribution of each dye in each detection window, can be determined. However, since the different spectral windows are measured in a time-sequential manner, any changes in the system in the time between the different measurements will result in errors. Such changes could for instance be due to variations in the excitation power.

In a PCR reaction, there can be more than 4 orders of magnitude abundance of one DNA strand over another. For example, assuming that strand A is present and results in maximum signal of 10000 AU, if there is a spectral cross talk of 10% with the spectral band of strand B this will result in a signal of 1000AU. If the excitation conditions (excitation power, detector gain, etc) changes by 1% when detecting the next channel this will result in an uncertainty of IOAU when detecting strand B. If the signal from strand B is very weak this weak signal can be completely overwhelmed by the varying contribution of signal A.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an improved analysis system and analysis method, which at least reduce one or more of the problems mentioned here before.

The invention is defined by the independent claims. The dependent claims define advantageous embodiments. According to the invention, there is provided an analysis system. This analysis system has a radiation source arrangement that provides input radiation that combines multiple radiation source frequency bands, that may be (but need not be) independently controllable, and multiple detectors each one of them having a multiple pass band filter, so that multiplexing can be implemented with a minimum number of movable components. The independent control of the input radiation frequency components means that one frequency component can be sent into a sample while the others are not. In this way, the light source arrangement has at least two different output radiation spectra, which can be provided in sequence. In general, the radiation used in the analysis system and method of the invention preferably is optical radiation, or light such as light of the UV and/or visible spectrum. Preferably the input radiation generates luminescence radiation within the sample, where luminescence radiation is understood to comprise fluorescence and or phosphorescence radiation. The sequential measurements can be taken quickly, improving the accuracy of the results with respect to variations in conditions in time and thus reducing problems as explained here above. In particular, quick switching of filters is provided since there is no mechanical switching of filters required. Thus it is possible to quickly cycle through the different spectral detection windows and obtain measurement results for all sample constituents to be detected. The result of measurements can be used to reduce the effect of cross talk between the different detection windows.

It is possible for the system to detect a number of excitation frequencies corresponding to the product of the number of detectors and the number of input radiation source frequency bands, and using a number of sequential measurements corresponding to the number of radiation source frequency bands.

The system of the invention enables a compact and cost effective method for multiplexed sample analysis, i.e. the detection of multiple constituents of a sample in one measurement run where each constituent is capable of providing a physical effect to be detected by the analysis system upon stimulation with a radiation source. The invention combines spectral and time sharing to keep the number of sources and detectors lower than the number of simultaneously/sequentially detected substances. In the known art, an increase in the number of simultaneously or sequentially detected labels leads to an increase of both the number of sources and detectors, or an increase in the number of filter pairs used. The present invention allows a compact and faster analysis system to be made. In addition the robustness of the analysis system will be improved, which in turn will improve reliability of the device. The device according to the invention is suitable for hand held analysis and when used in the field of diagnostics for point of care diagnostics, in settings outside the hospital environment.

In an embodiment, the radiation source arrangement comprises a plurality of switchable radiation sources and means for combining the radiation source outputs so as to form the input excitation radiation and providing the input radiation to a sample to be analyzed. Each radiation source can then be turned on or off independently, but without requiring mechanical components to be moved to route the radiation source output to the sample.

In an embodiment the means for combining the radiation source outputs comprise one or more dichroic mirrors. This results in a particularly robust and compact design of the analysis system. In an embodiment at least one beam splitter can be used for splitting the excitation- induced output radiation into output radiation portions, and routing each portion to a respective one of the plurality of detectors. The beam splitter advantageously allows splitting without moving parts.

In a preferred embodiment each frequency component of the excitation radiation preferably gives rise to excitation of a plurality of constituents of the sample. The excitation of multiple constituents with different excitation radiation frequency spectra then results in a set of measured detected radiation spectra from which a set of equations can be deduced for every constituent. Solving the equations enables determination of the component of each output radiation frequency. Moreover, this can be done using a reduced number of moving parts in the analysis system. For example, in an embodiment, there are two radiation sources, and each one of them gives rise to preferential excitation of at least two species in the sample, such that the system is for excitation of four species. In another example, there are three radiation sources, and each radiation source gives rise to preferential excitation of at least two species in the sample, such that the system is for excitation of six species. In an embodiment, the filter arrangement has at least two output radiation pass bands. In an embodiment each filter has three output radiation pass bands. The filters placed in front of the detectors have multiple wavelength ranges in which they transmit the output radiation. In an embodiment, the system further comprises a plurality of label species for introduction to the sample, the label species being capable of undergoing excitation at the frequencies of at least one of the multiple frequency bands. The label species may take any chemical or physical form as long as their output radiation is generated in response to the presence or absence of a specific constituent of the sample. In this embodiment, the constituents of the sample to be detected do not need to be capable of providing excitation output radiation as that will be provided by the label species instead in response to a specific recognition process, or absence thereof, of one of the constituents to be detected. Thus, labels may for example be incapable of undergoing excitation before having adhered to a specific constituent, or the other way around.

The term label substance herein is understood to include all entities that provide a signal that can be measured or detected by the analysis system upon illumination, wherein illumination may be excitation. Such labels include for example luminescent labels that are capable of fluorescence and/or phosphorescence after appropriate excitation. However any label that is capable of providing a different detectable effect, such as for example scattering and/or absorption of radiation, may be also well used, although luminescent labels and in particular fluorescent labels (also designated as fluorophores) are preferred, since fluorescence excitation is relatively efficiently generated and its detection very sensitive. The entities may be chemical entities such as molecules, clusters or complexes of molecules and/or biological species of any sort.

In an embodiment the radiation source arrangement comprises a laser diode or diodes. Such diodes provide relatively intense radiation, in specific desired frequency bands, while they are conveniently small. This is advantageous for e.g. the hand held variant of an analysis system according to the invention. However, any other source may alternatively be used as long as it provides the required input excitation radiation.

In a preferred embodiment of the analysis system, the excitation induced output radiation of the sample is fluorescence radiation and the sample comprises constituents that take part on an oligonucleotide replication process to be analyzed. The analysis system may comprise a polymerase chain reaction apparatus, for example a qPCR apparatus.

According to the invention there is further provided an analysis method. The method is advantageous on the same grounds as described for the analysis system herein above, e.g. the method provides multiplexed analysis with a minimum number of steps related to moving of filters and/or sources. According to the invention there is provided a computer program product. The processing step can be implemented in software and the software may be used to cause a programmable device to execute the method of the invention. For example the programmable device may be an electronic integrated circuit manufactured according to standard semiconductor industry methods. The integrated circuit may be part of a personal computer or a dedicated driving device.

BRIEF DESCRIPTION OF THE DRAWINGS

Examples of the invention will now be described in detail with reference to the accompanying drawings, in which:

Figs. Ia and Ib show schematically two techniques for generating fluorescence during DNA replication in a quantitative PCR technique;

Figs. 2a and 2b show the excitation absorption spectra and emission spectra for known fluorescent dyes used in a multiplexing optical analysis apparatus; Fig. 3 shows a first example of optical analysis apparatus of the invention;

Fig. 4 shows the dye emission spectra and filter band pass characteristics for the apparatus of Fig. 3 (and Fig. 5); and

Fig. 5 shows a second example of optical analysis apparatus of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

Nucleotide replication can be done making use of so called amplification techniques. One type of amplification technique involves the denaturing of double-stranded DNA at elevated temperature (typically > 90 degrees Celsius), specific binding of primers to the DNA sample at a reduced temperature (approximately 65 degrees) and copying of the original sequences starting from the primer position (at approximately 70 degrees). This procedure is repeated and in every cycle the amount of DNA with the specific sequence is doubled (when proceeding at 100% efficiency). The technique is well known in the art and often abbreviated as PCR meaning polymerase chain reaction.

After amplification, the presence of target DNA is detected by measuring the fluorescence intensity of the labeled amplified DNA, for instance after electrophoretic separation in a capillary or after hybridization to so-called capture probe substances which are for example applied immobilized in spots on a surface over which the amplification product is flowed. The surface may be part of a substrate that is included within a cartridge. These methods allow a qualitative answer of whether a certain target sequence was present in the sample or not.

For a quantitative determination of the concentration of target DNA the so- called quantitative (q-PCR) or real-time PCR technique is available. q-PCR is based on the general method of PCR amplification but it allows monitoring the DNA concentration dynamically, at the end of every amplification cycle. This is based on special fluorescent probes that luminesce only when hybridized to the amplified DNA product.

Several different approaches are known, and two are shown schematically in Fig. 1. In Fig. Ia, the CBRgreen fluorophore is represented by the circles. These are quenched, i.e. do not fluoresce, if not incorporated into a double-stranded DNA molecule, and become fluorescent when bound to a double-stranded DNA, as shown at 10. With increasing concentration of double-stranded DNA after every cycle of the PCR, the fluorescence signal will increase.

An alternative approach is the so-called Taq Man probe shown schematically in Fig. Ib. This technique is bound to a specific sequence of single-stranded DNA after denaturing. In this state, energy transfer to an adjacent dye quenches the fluorophore. When the DNA sequence is replicated starting from the primer, the probe is chopped and removed from the template as schematically shown therewith removing the dye from the vicinity of the fluorophore so that quenching is relieved. The amount of fluorescent signal is proportional to the amount of released fluorophores 12 that is proportional to the number of DNA molecules.

It is desired that the detection of fluorescence is required both for a qualitative and quantitative determination of the presence of a particular target DNA sample and for multiple sequences. In one embodiment of the invention the analysis system is an optical analysis system for detecting multiple excitation-induced outputs from a sample being analyzed. A light source generates an excitation light output comprising frequency components in multiple bands, with the frequency components independently controllable. A plurality of detectors is used to avoid the need to move filters, and a (fixed) filter arrangement is associated with each detector, with multiple pass bands. The filter arrangement can be a combination of filters or a single filter with the required multiple band pass characteristic. The detector signals can be processed to determine the levels of excitation- induced output from the sample at a plurality of frequencies greater in number than the number of detectors. The system can be used for exciting fluorescence in a sample, for subsequent detection as part of a biosensing procedure such as the replication process described hereinbefore.

Methods are known for the detecting fluorophores in a device by exciting the fluorophores by light radiation through an objective lens and collecting the luminescence, for example through the same lens in a reflective mode. The luminescent radiation is projected onto a sensor device after having passed a filter device to select the appropriate wavelength range. A confocal imaging arrangement is typically used.

In a known fluorescence scanner, a sample to be investigated is confined into a given volume. Light generated by a light source such as a laser is used to excite fluorescence. The light is collimated by a collimator lens and subsequently focused in the sample by means of an excitation lens.

The excitation lens can move relative to the sample, preferably in all three dimensions, to provide scanning across the sample so that the full sample can be analyzed. This relative motion can be decoupled arbitrarily, for example the sample can move in to the x-y plane and the lens in the z direction. Alternatively, the sample can be kept fixed and the lens has all the three-degree of freedom (x-y-z) on its own. Any other arrangement is also possible.

The induced fluorescence, (as a result of the excitation light focused into the sample) is collected by a collection lens, which can be the same component as the excitation lens, and is directed toward a detector.

Many different types of detector can be used such as a photon tube multiplier, avalanche photon detector, CCD detector or photodiode detector.

For confocal imaging, the excitation volume is kept to a minimum, ideally to the diffraction limited spot that the excitation lens can create. A typical confocal volume is in the order of a cubic micron, depending on the strength (numerical aperture, NA) of the excitation lens. The fluorescence created in this volume is collected by the collection lens and is imaged on the detector. In a confocal method, the focal point is confocal with a point in the detection path. At this point in the detection path, a small pinhole is typically placed to filter out any light coming from a location other than the focal point.

The light passing the pinhole is directed toward the detector. It is possible for the detector itself to play the role of the pinhole, with the restriction that the lateral size of the detector has to match the size of the focal point scaled by the numerical aperture of the imaging lens divided by the numerical aperture of the collection lens. This confocal mode is best suited to investigate a surface immobilization assay, as the result of an endpoint bio-experiment.

The invention concerns in particular the detection of multiple fluorescent species, namely from multiple dyes/labels introduced into the sample. A first embodiment of the invention is shown in Fig. 3, in the form of a PCR apparatus which operates essentially in the manner explained above. In this example, two lasers 101,102 are used in combination with two different detectors 701,702.

Standard off-the-self filters 601,602 can be used in combination with a set of standard dyes. Different, special purpose filters and dyes can of course also be used. One detailed example is given below.

The first laser 101 is laser diode with a wavelength around 650 nm (DL-4147- 162 from Sanyo). This light passes through a first dichroic mirror 201 (for example XF2016 (560DCLP) from Omega Opitical). This dichroic mirror is reflective for the light coming from the second laser 102 which is a 532nm diode pumped laser (for example FB532 from RgBlase).

The combined beam paths are reflected by reflector 501. This can either be a partly reflective mirror or a dichroic mirror (for example XF2054, from Omega Optical). The light is focused by lens 301 to the PCR chamber 400, and the generated fluorescence is collected by the same lens. This fluorescence light passes through element 501 and is then split by a 50-50 beam splitter 502. The light is directed towards the two different filters

601,602. A first channel contains filter 601 (XF3067 (515-600-730TBEM)) and detector 701. The other part of the light is filtered by filter 602 (XF3068 (495-575-700TBEM)) and focused on the detector 702.

The filters have multiple pass bands. In particular the filter 601 has pass bands centered on 515nm, 600nm and 730nm and the filter 602 has pass bands centered on 495nm, 575nm and 700nm.

To operate the system, the two laser outputs are provided in sequence. Each laser output excites the different dyes by different degrees. The detected filtered signals from both detectors can then be used to derive fluorescence levels for individual dyes as a set of simultaneous equations.

With this system, it is possible to excite and differentiate between up to four different dyes.

In this example, a number of different dyes from the ATTO series have been selected: the ATTO 550,590,647 and 700, other dyes are of course also possible. The spectra for the different dyes and the filters used are shown in Fig. 4. Fig. 4 also shows the spectra for two further dyes, ATTO 425 and 488 used in the second embodiment below.

Fig. 4 shows the multiple pass bands for the two filters. The emission by different labels is also shown in Fig. 4, and each label is mainly detected by one of the detectors, namely the detector having a filter band pass peak closest, or with most overlap with, the label emission frequency peak.

The first column in the Table 1 below shows the percentage of the emission at each label frequency which is allowed to pass by each filter. For example, the absorption of the 515-600-730 filter at the ATTO 550 frequency (which is a peak between approximately 500nm and 625nm as shown in Fig. 5) is 78%, so that 22% is allowed to pass. More of the peak is passed by the 495-575-700 filter, as the 575nm pass band is aligned more closely with the emission spectrum; 38% is allowed to pass.

Table 1

The second column converts the amount of light passed into percentages, for example for Atto 550, the ratio of 22:38 gives 22/60=37% and 38/60=63%.

Only the first two dyes (ATTO 550 and 590) are excited by the first laser at 532 nm. This means that when only this laser is on, any detected fluorescence comes either from either of these dyes. The signal on detector 701 will be dominated (65% vs 35%) by the fluorescence from the Atto 590 dye.

The signal from detector 702 will be dominated by the fluorescence from the ATTO 550 dye (63% vs 37%). By measuring both the total detected signal on both detectors and the ratio between the two signals it is possible to calculate the absolute signal from both dyes and thus their respective concentrations. It can be seen from the table above that the different dyes have a cross talk of about 50% into each others channel. This should however not cause a problem. By calibration, it is possible to differentiate between the contributions of the different dyes.

The arrangement does not require mechanical switching of any filters, and as a result it is possible to quickly switch between the different excitation lasers. Therefore, any changes between measurements of the different spectral windows will not result in problems in the calculation of the different dye concentrations.

A second embodiment is shown in Fig. 5, in which the same reference numbers are used as in Fig. 3. In this example, three lasers 101 , 102, 103 are used in combination with the same two different detectors as in the first example. Again, off-the-self filters are used in combination with a set of standard dyes.

The first laser 101 is the same 650 nm laser diiode and the output light passes through a first dichroic mirror 201 (again XF2016 (560DCLP) from Omega Opitical). This dichroic is reflective for the light coming from the second laser 102, which is the same 532nm diode pumped laser as in the first example. The combined light passes through the second dichroic mirror 202 (for example XF2027 (485DRLP) from Omega Optical). This mirror is reflective for the laser light from the third laser 103. This is a blue laser diode with a wavelength of 457nm (for example FB457 from RgBlase). Thus, the second embodiment adds the 457nm laser diode and its associated mirror 202, so that all three laser outputs are provided to the sample without the need for any mechanical switching.

In the same way as for the example above, the three combined beam paths are reflected by the element 501. The light is focused by lens 301 to the PCR chamber 400, and the generated fluorescence is collected by the same lens before routing to the two detectors 701,702.

With the system shown in Fig. 5, it is possible to excite and differentiate between up to six different dyes. As an example, the six dyes shown in Fig. 4 are used (the ATTO 425,488,550,590,647 and 700). Table 2 below corresponds to Table 1 above but adds the statistics for the additional two dyes. Table 2

It can be seen that the invention uses multiple excitation frequencies, for example from multiple lasers operating at different excitation wavelengths. Dichroic mirrors allow these different wavelengths to be combined to run along the same path.

The fluorescent light is split into two or more parts via a number of semi- reflective mirrors. The different separated fluorescence beams are led through different filters that allow a number of different spectral regions to pass through. Finally, the light is focused onto the detectors. These specific embodiments are only provided as way of example. Different combinations of lasers, labels and detection windows are of course possible. The detectors can be broad band detectors, with the selectivity introduced by the filters. The filters are multi-bandpass filters that transmit different parts of the spectrum. The different labels (dyes) are selected such that each has an absorption and emission spectrum such that, when the labels are sequentially excited by the different lasers, the contribution on the different detectors is significantly different.

The multiplex detection takes place by sequentially switching the lasers. Due to the differences in the absorption spectrum, the different lasers will excite a different subset of the labels. For each laser, the fluorescence signal on the different detectors is measured. The signal for the different labels will always fall to certain extent on multiple detectors as the filter responses have broad pass band peaks. However, by calibration in advance the different concentrations of the different labels can be determined as long as there at least as many quasi-independent measurements as there are labels. For N lasers combined with M detectors looking at the different spectral windows it is in principle possible to measure N x M different dyes with a sequence of N excitations.

Since there are no moving parts it is possible to rapidly switch the different lasers on and off, thereby reducing the problems usually encountered when different (spectral) windows are measured.

In all embodiments, the filters in front of the detectors should be selected such that they transmit multiple bands. The overlap between the transmission bands of one filter and with those of the other filters should be less than 50% preferably less than 10% most preferred less than 1%.

The filters in front of the detectors should strongly reduce the contribution of direct laser light preferably by at least a factor of 100 but more preferred a factor of 1000 but most preferred by a factor of more than 100000, so that the weaker fluorescence can be detected. It is important to select the spectra of the dyes carefully. For a maximum degree of multiplexing, it is preferred to select the dyes such that they fulfil the following criteria (assuming M detectors and N lasers):

For each of the N lasers a group of M dyes should be selected that are predominately excited by that laser. Preferably the excitation efficiency by that laser should be 10 times better than by the other lasers, more preferred more than 100 times better and most preferred more than a 1000 times better.

The M different dyes in such a group should have an emission spectrum such that the light falls predominantly on one of the M filter/detectors. It is preferred if the contribution of the dye on the "wrong" detector or detectors is less than 70% of that on the "correct" detector (i.e. at worst 59% vs 41%). It is more preferred if this contribution is less than 50%, most preferred if this is less than 25%

It is advantageous to rapidly switch between the different excitation lasers. One possible method is to use a block modulation driving scheme, wherein all the lasers are modulated with the same frequency somewhere between IHz and IMHz, more preferred between IkHz and 100KHz, with a duty cycle less than 50%. The phase of driving the different lasers is such that never more than 1 laser is on at the same time. The detection of the different detectors should be synchronous with the driving of the lasers. This ensures that the signals from the different spectral windows can be compared accurately. The invention can be applied to confocal or non-confocal systems, or indeed to systems which are able to switch between these different modes of operation, to provide combined endpoint and realtime PCR amplification and detection.

Some or all of the laser sources can be replaced by LED's or by a lamp with a filter to select the different excitation wavelengths.

The preferred applications of the invention are in the field of molecular diagnostics, clinical diagnostics, point-of-care diagnostics, advanced bio-molecular diagnostic research and optical biosensors, in particular related to DNA detection in combination with amplification methods, such as PCR, q-PCR, etc. The preferred application of the invention is thus in molecular diagnostics based on the detection of nucleic acids after amplification.

In the example above, the lens 301 is used both for the excitation light and the flurorescence light, and it can also be used for focus and tracking. Separate lenses may be used, for example with non-normal directions of illumination, or with operation in a transmissive mode. The focus and tracking is not shown in Figs. 3 and 5 to simplify the diagrams.

The excitation light can be generated by a single light source rather than separate lasers, if the output frequency can be switched.

The invention is in general applicable in the field of sample analysis wherein samples need to be examined volumetric or on a surface. The application of the invention may thus be in analytical methods requiring line excitation. These also include analysis on gaseous, liquid and/or solid samples.

Thus the invention may be used for chemical analysis of samples such as to determine their constitution or it may be used to inspect the evolvement or progress of a chemical or biochemical or biological process. Improved scanning speed enables the collection of more data points per time unit resulting in improved dynamic measurements. Without being limited to the field of bioanalysis, the preferred application of the invention is in the field of molecular diagnostics based on the detection of for example nucleic acids after amplification, proteins or other biochemical or biological entities. Further preferred fields of application include, clinical diagnostics, point-of-care diagnostics, advanced bio-molecular diagnostic research and optical biosensors, in particular related to DNA detection in combination with amplification methods, such as PCR, q-PCR, etc. The invention can also be used as a line scanner for imaging cells and/or tissue for example for pathology purposes. The can also be used for detection in an immunoassay to detect proteins. The above-mentioned embodiments illustrate rather than limit the invention, and at that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word "comprising" does not exclude the presence of elements or steps other than those listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that the combination of these measures cannot be used to advantage.

Summarizing, an analysis system detects multiple radiation outputs from a sample being analyzed. A radiation source generates input radiation comprising frequency components in multiple frequency bands, with the frequency components being independently controllable. A plurality of detectors are used and a (fixed) filter arrangement with multiple pass bands is associated with each detector. The detector signals can be processed to determine the levels of output radiation from the sample at a plurality of frequencies greater in number than the number of detectors, without having to move any filters.

The invention combines spectral and time sharing to keep the number of sources and detectors lower than the number of simultaneously/sequentially detected substances. This may be advantageously applied One example of multiplexed sample analysis is Real Time PCR with multiplexing of at least 2 different labels. Normally, an increase in the number of simultaneously or sequentially detected labels leads to an increase of both the number of sources and detectors, or an increase in the number of filter pairs used. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word "comprising" does not exclude the presence of elements or steps other than those listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that the combination of these measures cannot be used to advantage.

Claims

CLAIMS:

1. An analysis system comprising: a radiation source arrangement ( 101 , 102, 103) for generating input radiation to be provided to a sample, the input radiation comprising independently controllable frequency components in multiple frequency bands, - a plurality of detectors (701 ,702) for generating a plurality of detector signals, each respective one of the plurality of detectors being arranged for detecting an output radiation portion from the sample to generate one of the plurality of detector signals, the output radiation being generated by an interaction of the input radiation with the sample; a plurality of filter arrangements (601,602), each respective one of the plurality of filter arrangements having multiple pass bands and being arranged for filtering the portion of the output radiation to be detected by a respective one of the plurality of detectors (701,702); and means for processing the plurality of detector signals to determine the levels of output radiation from the sample at a plurality of frequency bands greater in number than the p lurality o f detectors .

2. The analysis system as claimed in claim 1, wherein the radiation source arrangement (101,102,103) comprises: a plurality of switchable radiation sources each of which is arranged for providing an input radiation component, and means (201,202) for combining the input radiation components to form the input radiation.

3. The analysis system as claimed in claim 2, wherein the means for combining the input radiation components comprise one or more dichroic mirrors (201,202).

4. The analysis system as claimed in any preceding claim, further comprising at least one beam splitter (501,502) for splitting the output radiation into the output radiation portions and routing each of them to a respective one of the plurality of detectors (701,702).

5. The analysis system as claimed in any preceding claim, wherein each frequency component of the input radiation is arranged to give rise to excitation of a plurality of constituents of the sample (400).

6. The analysis system as claimed in any preceding claim, wherein each filter arrangement (601,602) has at least two pass bands.

7. The analysis system as claimed in any preceding claim, further comprising a plurality of label species for introduction into the sample, the label species being capable of undergoing excitation at the frequencies of at least one of the multiple frequency bands.

8. The analysis system as claimed in any preceding claim wherein the output radiation comprises luminescence.

9. An analysis method comprising: generating (101 , 102, 103) an input radiation comprising independently controllable frequency components in multiple frequency bands, providing the input radiation to a sample to generate an output radiation by an interaction of the input radiation with the sample; filtering each one of a plurality of output radiation portions using a filter arrangement having multiple radiation pass bands; detecting each of the plurality of filtered output radiation portions using a respective one of a plurality of detectors (701.702) to generate a plurality of detector signals; - processing the plurality of detector signals to determine the levels of output radiation from the sample at a plurality of frequency bands greater in number than the plurality of detectors.

10. A computer program product for enabling a programmable device to carry out the method of claim 9.