DE102010010741A1 - Optical fiber device for emitting and receiving light, system, method and computer program product - Google Patents

Abstract

In summary, the present invention relates to a light guide device for emitting and receiving light, in particular for exciting and observing fluorescence, with - a light guide bundle fixation device and with - at least one light guide bundle, wherein the at least one light guide bundle is fixed by means of the light guide bundle fixation device and can be heated by means of the light guide bundle fixation device, a system, a method and a computer program product.

Description

The present invention relates to a temperature-controllable optical waveguide device for emitting and receiving light, a system for exciting and measuring fluorescence on at least one liquid receiving area, a method for the qualitative and / or quantitative detection of target molecules in a sample to be analyzed, and a computer program product.

State of the art

Optical systems are used in the field of laboratory diagnostics and medical research for the quantitative and qualitative determination of target molecules in patient samples. A variety of such optical systems, such as systems for photometric absorption measurement and fluorescence analysis are already known.

In the commonly performed fluorescence-based methods for the detection of nucleic acids fluorescent labeling molecules are added to a sample to be analyzed. One distinguishes between unspecific marking method z. Example by means of fluorescent DNA intercalators and sequence-specific detection methods in which the desired nucleic acid complementary DNA or RNA probes are used.

In general, excitation of the label molecules by means of light of predetermined wavelength and detection of the fluorescence emitted by the fluorescent label molecules enables the qualitative determination of a possible chemical reaction and, by evaluating the measured light intensities, also a quantitative analysis of this reaction.

The Polymerase Chain Reaction (PCR) is a procedure for DNA amplification with a cyclic character. By means of this reaction, which is highly relevant for practice, even the smallest amounts of a nucleic acid molecule optionally present in the sample can be amplified. In the meantime, the use of fluorescently labeled primers and probes for the simple fluorescence-based qualitative and quantitative detection of DNA has become widespread in PCR and other DNA amplification reactions.

In view of current developments in the field of DNA analysis, it is of increasing scientific as well as economic interest to reduce as much as possible the amounts of starting material used and the reaction volume for the amplification. This development has already progressed to the use of individual cells as starting material. The sample volumes used in the single cell analysis amount to only a few microliters. Processing such small sample volumes places a special requirement on a system for determining fluorescence in chemical reactions.

In many of these systems for the qualitative and quantitative measurement of fluorescence volumes of about 50 microliters to a few milliliters are usually used for the sample material to be analyzed. In addition, the starting material is usually provided in small sample tubes or in wells of multi-well plates, which, however, is unsuitable for the field of single-cell amplification. Likewise, manipulation of the sample during amplification and fluorescence measurement is not possible since these containers must remain closed during the amplification phase.

When using liquid samples, it has proven to be problematic that products evaporating from the sample can form deposits on the surfaces of optical measuring devices, which, in addition to reduced sensitivity, can also cause measurement errors in the determination of the fluorescence. This fouling effect is all the more serious if the liquid samples are additionally heated above ambient temperature during the analysis.

Object of the invention

It is the object of the invention to provide an improved apparatus as well as a system and method for exciting and / or observing fluorescence in liquid or wet samples contained in unclosed sample containers with the samples and apparatus in one closed volume and contamination of the device during the implementation of the method should be avoided or at least reduced.

This object is achieved according to the independent claims. Advantageous embodiments are the subject of the dependent claims.

Optical fiber device according to one aspect

One aspect of the present invention relates to an optical fiber device for emitting and receiving light, in particular for excitation and observation of fluorescence, with a Lichtleiterbündelfixierungsvorrichtung and with at least one optical fiber bundle, wherein the at least one optical fiber bundle is fixed by means Lichtleiterbündelfixierungsvorrichtung and heated by the Lichtleiterbündelfixierungsvorrichtung.

According to one aspect of the invention, the contamination of the optical fiber bundle during the excitation and observation of fluorescence by tempering the optical fiber bundle by means of Lichtleiterbündelfixierungsvorrichtung is avoided or reduced. Pollution of the optical fiber bundle is understood as meaning, for example, the condensation of molecules or molecular aggregates on the optical fiber bundle. These molecules or molecular aggregates can be obtained, for example, by evaporating wet or liquid samples, e.g. B. arise during the excitation and observation of fluorescence. The evaporated molecules or molecular aggregates are distributed in the closed volume in which sample and device are located. A tempering of the optical fiber bundles avoids or reduces the condensation of the molecules or molecular aggregates on these.

In particular, in the excitation and observation of fluorescence in heated liquid or wet samples, a corresponding heating of the optical fiber bundles of the optical fiber device effectively prevent condensation of liquid on the optical fiber bundles and thus ensure the undisturbed excitation and observation of fluorescence.

The term fluorescence is understood to mean the spontaneous emission of light during the transition of an electronically excited system into a state of lower energy. Certain substances may, for. B. be excited by irradiation with light to emit fluorescence. As a rule, the emitted light is lower in energy than the light previously absorbed by these substances.

An optical waveguide device according to the invention comprises at least one optical fiber bundle. Each optical fiber bundle is composed of at least two optical fibers that are suitable for the transmission of optical signals together. The at least one optical fiber bundle can be fixable spatially with respect to a sample to be observed by means of a heatable Lichtleiterbündelfixierungsvorrichtung. The light guides according to the invention are preferably glass fibers and / or polymer optical fibers with a diameter between 10 micrometers and 500 micrometers, preferably between 20 and 250 micrometers, and more preferably with a diameter of about 50 micrometers.

The at least one optical fiber bundle of the optical fiber device is fixed in the Lichtleiterbündelfixierungsvorrichtung.

In one embodiment, the optical fiber fixing device is designed as a continuous, preferably metallic plate. It has in an advantageous grid on several continuous openings, which are just so large that they each receive and fix a fiber optic bundle.

In a further embodiment, the optical fiber fixing device can also consist of more than one part or element. Thus, the individual optical fibers of the optical fiber bundle can be fixed, for example, first by a thermally conductive holder. This version consists for example of a metallic material. You can z. B. be executed as a known to those skilled crush detection. The optical fiber bundle fixing device according to this embodiment comprises a plurality of such sockets and a thermally conductive plate, which in an advantageous grid has a plurality of continuous openings for receiving in each case a socket.

For example, the thermally conductive sockets may protrude beyond the sample side surface of the thermally conductive plate toward the sample to be observed, preferably 2 to 20 mm, more preferably 3 to 15 mm, and most preferably about 7 mm. This is advantageous in order to influence the temperature of the sample to be observed as little as possible by the temperature control of the Lichtleiterbündelfixierungsvorrichtung.

Embodiments of the light guide device

According to one embodiment, the optical fiber fixing device comprises at least one heating device.

Such a heater, which consists at least partially of a heat-conducting, preferably metallic material may, for. B. be designed as electrical resistance heating, Peltier element or as a tempered liquid flow-through heating element.

In one embodiment of the light guide device, the light guide bundle fixing device is arranged on an end region of at least one optical fiber bundle.

On the one hand, this enables a precise placement and, on the other hand, a targeted temperature control of the end region of the at least one optical fiber bundle of the optical waveguide device relative to a sample to be observed.

The sample-side end region of an optical fiber bundle, in other words the sample-side optical fiber bundle end region, is understood to be the region of the optical fiber bundle which is directly surrounded by the heatable optical fiber bundle fixing device. The corresponding end surface of the fiber optic bundle is sealed in The following is referred to as the sample-side end surface of an optical fiber bundle or, in other words, as the sample-side optical fiber bundle end surface.

At the sample-side end surface, optical signals can be recorded in or emitted from the at least one optical fiber bundle. The ends of the individual optical fibers of an optical fiber bundle are arranged substantially flush with one another in its end region. In other words, all optical fibers of an optical fiber bundle terminate substantially at the same height in the end region of an optical fiber bundle, so that the end surfaces of the optical fiber bundles are substantially planar.

According to one embodiment, the Lichtleiterbündelfixierungsvorrichtung is designed as a heater.

Consequently, the Lichtleiterbündelfixierungsvorrichtung and in particular the end portion of the at least one optical fiber bundle can be tempered without the aid of an additional heating device. The Lichtleiterbündelfixierungsvorrichtung can in this embodiment, for. B. act as electrical resistance heating or be an electrical resistance heater.

According to one embodiment, the optical waveguide device has at least one optical fiber bundle which comprises at least one excitation optical fiber bundle with at least one excitation optical waveguide and at least one emission optical waveguide with at least one emission optical waveguide.

By way of example, the excitation light guides of the at least one excitation light guide bundle of the light guide device run in such a way that light from a light source can be guided onto a sample to be observed by means of the excitation light guide bundle. By contrast, the emission light guides of the at least one emission light guide bundle of the light guide device run in such a way that light from the sample to be observed can be guided, for example, to a detector. An optical fiber bundle comprises at least one excitation optical fiber bundle and at least one emission optical fiber bundle. The excitation optical fiber bundles and emission optical fiber bundles are brought together at the sample-side end region of the optical fiber bundle facing the sample to be observed. The excitation optical fiber bundles and emission optical fiber bundles are fixed there by means of the optical fiber bundle fixing device in such a way that a sample to be observed can both be illuminated by the optical fiber bundle and light emitted by this specimen can be received in the optical fiber bundle.

Advantageously, the excitation light guide and the emission light guide are homogeneously mixed at the end region of the light guide bundle so that the number of excitation light guides and emission light guides are approximately the same in each region of the cross section of the light guide bundle end region. This ensures a homogeneous delivery of light by means of the optical fiber bundle to a sample to be observed and a homogeneous absorption of light by means of the optical fiber bundle starting from a sample to be observed.

According to one embodiment, excitation optical fiber bundles or emission optical fiber bundles comprise 1 to 5000, preferably 1 to 1000, particularly preferably 1 to 500 and most preferably 10 to 300 excitation light guides, or 1 to 5000, preferably 1 to 1000, particularly preferably 1 to 500 and most preferably 10 up to 300 emission light guides. For example, the excitation fiber bundles and emission fiber bundles of an optical fiber bundle may contain approximately the same number of excitation optical fibers and emission optical fibers. A corresponding optical fiber bundle can thus comprise, for example, in each case 50 excitation light guides and emission light guides, which are uniformly mixed in the region of the cross section of the optical fiber bundle end region.

In one embodiment variant, the at least one optical fiber bundle comprises more emission light guides as excitation light guides.

Preferred ratios between excitation light guides and emission light guides are between 1: 1.1 and 1:10, preferably between 1: 1.5 and 1: 5 and more preferably at about 1: 2.

By providing more emission light guides relative to excitation light guides, the sensitivity of the fluorescence measurement device can be increased without significant losses in fluorescence excitation.

According to one embodiment, the light guide device comprises at least one light source and at least one detector, wherein the at least one excitation light guide bundle is associated with the at least one light source and at least one detector is associated with the at least one emission light guide bundle.

In other words, the at least one light source may be spatially associated with the at least one excitation optical fiber bundle. The at least one detector may be spatially associated with the at least one emission light guide bundle.

The at least one light source comprises, for example, a laser, a laser diode, an LED, a gas discharge lamp, and / or an incandescent lamp.

By way of example, the optical waveguide device may comprise the at least one light source and the at least one detector in addition to the optical fiber bundle, wherein the optical fiber bundle comprises at least one excitation optical fiber bundle and at least one emission optical fiber bundle.

This assignment advantageously has the effect that light from at least one light source can be conducted via the at least one excitation light bundle to at least one sample to be observed and light emitted from this sample via the at least one emission light bundle to at least one detector by means of the light guide device. Likewise, light from a plurality of light sources can be directed simultaneously via several excitation light guide bundles to one or more samples to be observed, and from there can be conducted simultaneously to a plurality of detectors via a plurality of emission light guide bundles.

According to one embodiment, the at least one optical fiber bundle comprises N excitation optical fiber bundles, to which N light sources are assigned, and M emission optical fiber bundles, which are assigned to a common detector. The variables N and M are each a natural number with values between 2 and 1000, preferably between 5 and 500, more preferably between 10 and 250 and most preferably with the value 48. The variables N and M can have different values. The variables N and M can have identical values.

For example, a detector may be implemented as a photomultiplier tube, avalanche photodiode, photodiode, CCD or CMOS chip, or generally in the form of any light-sensitive substance, or may comprise a photodiode, a CCD or CMOS chip, or generally any light-sensitive substance. The detector can be read out by means of a read-out device during operation of the light guide device.

According to one embodiment, the detector is temperature-controlled.

The detector can be heated and / or cooled using a suitable tempering element in the operation of the light guide device in a range of 5 ° C to 60 ° C, preferably 15 ° C to about 35 ° C. During operation of the optical waveguide device, it is particularly preferred to keep the temperature of the detector substantially constant. In other words, the detector is tempered such that its setpoint temperature does not differ from its actual temperature within a certain period of time by more than 5 ° C., preferably not more than 2 ° C., and more preferably not more than 1 ° C. As Temperierungselemente come, for example, Peltier elements, based on temperature-controlled liquids and / or air mass heating or cooling technologies or facilities in question. Such a detector allows, for example, the setting of a substantially constant temperature at the detector to compensate for changes in the ambient temperature, which could result in a falsification of the measurement results.

System according to one aspect

One aspect of the invention relates to a system for exciting and measuring fluorescence on at least one liquid receiving region with a light guide device according to the invention, a substrate whose surface comprises at least one liquid receiving region, and with a substrate tempering device, wherein the substrate is tempered by means of the substrate temperature control device and the optical fiber device relative is arranged to the substrate that the at least one optical fiber bundle of the optical fiber device and the substrate are non-contact.

A substrate in the sense of the present description may be an article which is thermally conductive and on whose surface samples for fluorescence analysis can be positioned. Glass and / or plastic slides may be exemplary substrates. The glass and / or plastic slide may, for example, be essentially cuboid, wherein one surface of the cuboid substrate may be the surface on or on which one or more samples for fluorescence analysis can be positioned.

According to the invention, the term liquid-receiving region means an area of the surface of a substrate on or on which a liquid to be observed can be specifically positioned. Such liquid receiving areas according to the invention can be, for example, superficial cavities and / or liquid receiving areas obtained by hydrophobic and / or hydrophilic modification of the surface structure. For example, a plurality of liquid receiving areas are provided on the substrate, which are separated from each other, for example, by suitable elevations of the substrate surface and / or by hydrophobic coatings of the substrate surface. Systems according to the invention having a plurality of liquid receiving areas preferably have 2 to 1000, more preferably 10 to 250 and most preferably 48 liquid receiving areas. It is also possible to arrange 2, 3, 4, etc. substrates which, in each case or together, have 2 to 1000, particularly preferably 10 to 250 and most preferably 48 liquid receiving areas.

An exemplary Substratemperierungsvorrichtung allows the variable temperature of the substrate or substrates and the samples positioned thereon to be observed. An exemplary substrate temperature control device may comprise any desired heating device and any desired cooling device. For efficient heating or cooling of the substrate by means of the substrate temperature control device, a sufficient thermal coupling between the substrate and the substrate temperature control device is provided, which, for. B. can be ensured by the largest possible contact between Substratemperemperierungsvorrichtung and substrate. When tempering planar or planar substrates, a substrate tempering device accordingly comprises a planar surface. This surface or surface may be suitable or designed for positioning and temperature control of the substrate. In a particular embodiment of the substrate temperature control device, a planar receptacle for positioning and temperature control of the substrate is provided on one of the outer sides of the substrate, preferably on the upper side of the substrate. The top can also be referred to as top surface or surface. In the operating state of the system, the substrate is arranged such that the upper side of the substrate and the Lichtleiterbündelendfläche the associated at least one optical fiber bundle are opposite. In other words, end faces of the excitation light guides through which light is emitted to the substrate and end faces of the emission light guides through which light from the sample is received in the emission fibers may be arranged substantially parallel to the upper surface of the substrate. Under parallel in this sense is also to be understood that depressions, bumps, etc. may be present on the substrate and, for example, remain between the depressions, bumps, etc. planar surface areas of the top. The planar surface areas of the upper surface of the substrate and the end surfaces of the excitation light guides and end surfaces of the emission light guides may be substantially parallel.

Possible embodiments of such a substrate temperature control device, for example, provide devices for carrying out the polymerase chain reaction (PCR), such as. B. the device TC4000 / FTC4 / Flat of the company Techne or the device T1 thermal cycler with in-situ block of the company Biometra, is particularly suitable for tempering planar or planar substrates z. B. the use of the device "AmpliSpeed" Beckman Coulter Biomedical GmbH (Munich, Germany) proved. Thus, one aspect of the invention may also include the use of a light guide device according to the invention in or with a substrate tempering device, in particular in conjunction with a cuboid substrate.

The system according to the invention for exciting and measuring fluorescence on at least one liquid receiving region is designed such that the at least one optical fiber bundle of the optical fiber device and the substrate are positioned without contact relative to one another such that a gap filled with gas is formed between the at least one optical fiber bundle and the substrate.

In one embodiment, all the optical fiber bundles of the optical fiber device and the substrate are positioned corresponding to each other without contact. The intermediate space advantageously prevents contamination of the at least one optical fiber bundle with liquid samples applied to the substrate. Contamination of the system when successively examining multiple samples on different substrates, d. H. when changing the substrates, can thus be effectively avoided.

Embodiments of the system

In one embodiment of the system, the at least one optical fiber bundle fixed by the optical fiber fixing device ends above the substrate, and the shortest distance between the end surface of the at least one optical fiber bundle and the substrate is between 0.1 mm and 20 mm, preferably between 0.5 mm and 10 mm , more preferably between 1 mm and 5 mm, and most preferably 3 mm.

The term 'above' in this context means that the distance between the substrate surface and the end face of the at least one optical fiber bundle is adjusted along the perpendicular to the sample-side surface of the substrate. This also applies if the substrate has depressions, unevenness, etc. and, for example, flat surface areas remain between the depressions, unevennesses, etc. The distance between the sample-side substrate surface and the optical fiber bundle end surface may be set along the normal to the planar surface areas of the surface of the substrate and the optical fiber bundle end surface. In other words, the distance is the distance along the normal between the optical fiber bundle end surface and the planar surface regions of the surface of the substrate.

The setting of an optimal distance between the substrate surface and the end of the Fiber optic bundle to ensure that contamination of the fiber optic bundle ends, ie the Lichtleiterbündelendflächen is avoided with sample liquids or sample material. At the same time, a sufficient amount of light can be emitted to excite fluorescence from the at least one optical fiber bundle end face, the sample being positioned in a region of the substrate surface to be illuminated. Furthermore, a sufficient amount of light for measuring the specific fluorescence of fluorescent samples on the substrate surface can be picked up by the optical fiber bundle end face. The recording of nonspecific fluorescence, which in contrast to the above-mentioned specific fluorescence does not originate from the sample illuminated with the optical fiber bundle, but for example from an adjacent sample, into the optical fiber bundle can be avoided according to the described embodiment by the choice of a suitable distance of the optical fiber bundle end to the substrate ,

In this case, the distance to be used of the fiber optic bundle end to the substrate depends on how large the surface of the substrate to be illuminated by an optical fiber bundle, z. B. exactly one liquid receiving area, is. It is likewise dependent on in which maximum exit angle or acceptance angle α the light emerges from the optical fiber bundle relative to the optical axis of the optical fiber bundle and at what distance optionally a plurality of regions to be observed independently are provided on the surface of the substrate.

a

der Abstand zwischen dem Lichtleiterbündelende und einem zu beobachtenden Bereich auf der Substratoberfläche ist. Der Abstand a kann auch als idealer Abstand bezeichnet werden.

α

der maximale Austrittswinkel des Lichts aus dem Lichtleiterbündel relativ zur optischen Achse des Lichtleiterbündels ist.

d

der mittlere Durchmesser der Lichtleiterbündelendfläche ist.

D

der mittlere Durchmesser des zu beobachtenden Bereichs auf der Substratoberfläche ist.

For example, the cone of light emerging from an optical fiber bundle neither illuminates substantially more nor substantially less than the area of the substrate surface to be observed, for example precisely one liquid absorption region or a defined subregion of a liquid absorption region. In this case, the distance between the optical fiber bundle end and a region to be observed on the substrate surface is given by the following formula: a = (D - d) / (2 · tan α) [Formula 1] in which:

a

is the distance between the fiber optic bundle end and an area to be observed on the substrate surface. The distance a can also be called the ideal distance.

α

the maximum exit angle of the light from the optical fiber bundle is relative to the optical axis of the optical fiber bundle.

d

is the mean diameter of the optical fiber bundle end face.

D

is the mean diameter of the area to be observed on the substrate surface.

The maximum exit angle α may be dependent on the material of the optical fiber used and is z. B. in the case of stepped fibers usually in a range between 11.5 ° and 25.5 °.

The distance between the optical fiber bundle end surface and the substrate surface may also differ by up to 50% from the ideal distance a to be determined according to formula 1. However, it is by no means less than the maximum height of a liquid or wet sample applied to the substrate surface in order to prevent soiling of the optical fiber bundle end surfaces. When observing liquid or moist samples by means of tempered fiber bundle ends, a sufficient distance between the liquid sample to be observed and the tempered fiber bundle end must also be maintained in order to reduce unwanted temperature influences on the sample.

As with increasing distance of the Lichtleiterbündelendfläche to the substrate increased scattered light and / or nonspecific fluorescence, z. B. of adjacent liquid receiving areas, in the Lichtleiterbündelendfläche can occur, the preferred distance range between the Lichtleiterbündelendfläche and the substrate is also limited upwards. If a plurality of liquid receiving regions are to be observed on the substrate independently of one another by means of a plurality of optical fiber bundle ends, the preferred distance of the corresponding optical fiber bundle end surfaces from the substrate is also determined by the distance of these liquid receiving regions from one another on the substrate. The denser the liquid receiving regions to be observed lie on the substrate, the lower the preferred distance of the corresponding optical fiber bundle end surfaces from the substrate.

In one embodiment, the surface of the at least one liquid receiving area has modified wetting properties and comprises a central, preferably circular hydrophilic reaction area, a preferably annular hydrophobic central area surrounding the reaction area and a preferably annular hydrophilic outer area surrounding the central area. The outer area is in turn surrounded by a hydrophobic area.

The substrate surface in the region of such a liquid receiving region is preferably substantially planar or planar. Particularly preferred are completely planar or plane Substrates with multiple liquid receiving areas, for example 7.5 cm long and 2.5 cm wide glass or plastic slides with 48 liquid receiving areas. When using substrates having a plurality of hydrophobically structured liquid receiving areas, the areas between the individual liquid receiving areas advantageously have a hydrophobic surface structure.

The reaction area is preferably arranged in the center of the respective liquid receiving area. It serves to apply a sample and / or reaction liquid to be observed, which is prevented from flowing on the surface by the hydrophobic, preferably annular hydrophobic region surrounding the reaction region. For protection against evaporation, liquid sample and / or reaction liquids positioned on the reaction zone are advantageously covered with an oily liquid. The hydrophobic substrate surface surrounding the annular hydrophilic outer region of the liquid-receiving region prevents bleeding of the oily liquid from the region of the liquid-receiving region. The above statements apply mutatis mutandis to a large number of liquid intake areas for each liquid intake area.

When using substrates with liquid receiving regions which have wetting properties modified in this way, the optimum distance a between the at least one fiber optic bundle end surface and the substrate surface is preferably set so that the light emerging from the fiber optic bundle end surface strikes substantially exclusively the central, preferably circular, hydrophilic reaction region. so that essentially only the fluorescence occurring in the reaction region is measured. The area D to be observed on the substrate surface in this case corresponds to the mean diameter of the hydrophilic reaction area of the liquid wetting areas with modified wetting structure. In compliance with the above-mentioned restrictions, the optimum distance a between the end of the optical fiber bundle and the substrate surface (according to formula 1) can also be exceeded or fallen below by up to 50%.

In one embodiment of the system for exciting and measuring fluorescence on at least one liquid receiving area, at least one liquid receiving area is assigned an optical fiber bundle end fixed by the optical fiber fixing device, the liquid receiving area and the optical fiber bundle end area of the associated optical fiber bundle having substantially the same dimensions and the liquid receiving area and the associated optical fiber bundle being so aligned with each other, that from the Lichtleiterbündelendfläche emerging light strikes the associated liquid receiving area.

When using liquid receiving regions which have a reaction region as part of the liquid receiving region, for example, the reaction region of the liquid receiving region and the optical fiber bundle end surface of the associated optical fiber bundle can have substantially the same dimensions. In other words, the reaction area of the liquid receiving area and the associated optical fiber bundle end face have a substantially identical diameter, or, for non-circular areas, a substantially identical mean diameter.

In the context of this description, the criterion of essentially identical dimensions should also be met if the average diameter of the liquid receiving region or the average diameter of the reaction region of the liquid receiving region is 0.25 to 4 times the average diameter of the optical fiber bundle end surface of the associated optical fiber bundle end , Since light emerging from the optical fiber bundle end is scattered relative to the optical axis of the optical fiber bundle, it is advantageous if the average diameter of the liquid receiving region is slightly larger than the average diameter of the optical fiber bundle end surface of the associated optical fiber bundle end.

For example, in one embodiment, the circular reaction area of a liquid receiving area has a diameter of 1.6 mm, while the diameter of the optical fiber bundle end area of the associated optical fiber bundle end is 1 mm. By setting a distance of about 3 to 3.5 mm between the optical fiber bundle end and the substrate can be ensured when using the above-mentioned dimensions that light emerging from the Lichtleiterbündelendfläche substantially only on the reaction region of the liquid receiving area and effective from this outgoing fluorescence with the associated fiber optic bundle end can be detected.

For example, a liquid receiving area and a fiber optic bundle end are each associated with each other such that the center of the liquid receiving area and the center of the Lichtleiterbündelendfläche the associated Lichtleiterbündelendes come to rest on a common perpendicular to the substrate surface. The liquid receiving region and the associated optical fiber bundle end can also be displaced relative to one another parallel to the substrate surface, but only so far as to extend through the center of the optical fiber bundle end surface of the optical fiber bundle end going normal to the substrate surface would cut the substrate in the area of the liquid pickup area, preferably in the area of the reaction area of the liquid pickup area. The optical fiber bundle ends are preferably aligned with the substrate in such a way that the light exiting from the center of the optical fiber bundle end surface strikes the surface of the substrate facing the optical waveguide device substantially perpendicularly, but maximally at an angle of 60 °.

Method according to one aspect

• – einen Probenbereitstellungsschritt, wobei eine zu analysierenden Probe auf zumindest einem Liquidaufnahmebereich auf einem Substrat bereitgestellt wird,
• – einen Substrattemperierungsschritt, wobei das Substrat mittels einer Substrattemperierungsvorrichtung temperiert wird,
• – einen Lichtleiterbündeltemperierungsschritt, wobei zumindest ein Lichtleiter mittels einer Lichtleiterbündelfixierungsvorrichtung temperiert wird,
• – einen Belichtungsschritt, wobei die zumindest eine zu analysierende Probe mittels einer Lichtleitervorrichtung belichtet wird,
• – einen Detektionsschritt, wobei von der zu analysierenden Probe ausgehendes Licht mittels der Lichtleitervorrichtung empfangen wird, und
• – einen Auswertungsschritt, wobei das im Detektionsschritt empfangene Licht zum qualitativen und/oder quantitativen Nachweis der gegebenenfalls in der zu analysierenden Probe vorhandenen Zielmoleküle verwendet wird.

A further aspect of the invention relates to a method for qualitatively and / or quantitatively detecting target molecules in a sample to be analyzed, in particular using a system according to the invention, comprising:

• A sample providing step, wherein a sample to be analyzed is provided on at least one liquid receiving area on a substrate,
• A substrate tempering step, wherein the substrate is tempered by means of a substrate tempering device,
• A Lichtleiterbündeltemperierungsschritt, wherein at least one optical fiber is tempered by means of Lichtleiterbündelfixierungsvorrichtung,
• An exposure step, wherein the at least one sample to be analyzed is exposed by means of an optical waveguide device,
• A detection step, wherein light from the sample to be analyzed is received by means of the light guide device, and
• An evaluation step, wherein the light received in the detection step is used for the qualitative and / or quantitative detection of the target molecules optionally present in the sample to be analyzed.

Qualitative detection serves to detect the presence or absence of target molecules, while quantitative determination can provide information on the number or frequency of target molecules in the observed sample. In the context of this description, target molecules may be any inorganic or organic molecules which either have fluorescent properties themselves or can be labeled by means of fluorescent dyes. Fluorescence-labeled molecules are detected by detection of the fluorescent dye used for labeling. Examples of possible target molecules are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), proteins and / or any other metabolites and products of cellular metabolism.

In the sample providing step, at least one liquid or wet sample containing target molecules to be assayed is positioned on a liquid receiving area, preferably on the reaction area of a liquid receiving area. Depending on the analysis to be carried out, it is possible to provide either fluorescence samples or non-fluorescent samples which are labeled with a fluorescent dye only later in the process but before the detection step. It is also possible to provide both fluorescent and non-fluorescent samples on a substrate.

For tempering the substrate, this is brought into thermal contact with the substrate temperature control device in the substrate tempering step and brought to at least one desired temperature at least once. The temperature control can be a cooling, a heating or a constant temperature control of the substrate. Constant temperature control means in this context that the substrate is heated so that its target temperature of the actual temperature within a certain period of time by not more than 5 ° C, preferably not more than 2 ° C and more preferably not more than 1 ° C is different.

Before, after or at the same time as the substrate tempering step, the optical fiber bundle tempering step is carried out, wherein the optical fiber bundle ends are tempered by means of the optical fiber bundle fixing device to a constant or sequentially to several different desired temperatures. The optical fiber bundle ends can be heated, cooled or kept at a constant temperature in this step. The optical fiber bundle ends can be heated and / or repeatedly cooled several times in this step and / or kept at a constant temperature between heating and / or cooling steps. If the substrate is heated in the substrate tempering step, heating of the fiber bundle ends can also be carried out, for example, in the optical fiber bundle tempering step. According to the invention, a condensation of evaporating sample liquid on the optical fiber bundle ends and thus an impairment of their optical properties is reduced, in particular avoided. If the heated optical fiber bundle ends do not significantly influence the substrate temperature by the emission of heat, the optical fiber bundle ends can also be kept at a temperature above the substrate temperature in a substrate tempering step to simplify process control during any cooling phases that may be present.

During the exposure step, by means of the light guide device light from a Lichtleiterbündelendfläche radiated on at least one sample to be analyzed. This can be done once or several times at any time interval.

During and / or after the exposure step, the fluorescence emanating from the sample to be analyzed is recorded in the detection step by means of at least one optical fiber bundle and passed via the optical fiber device to at least one detector which detects the type and / or intensity of the fluorescence as fluorescence information. In the context of the method according to the invention, any number of detection steps can be carried out, preferably in each case during an exposure step. Before and / or after an exposure step, if appropriate, a measurement of the background signal can also be carried out. This is advantageous in order to be able to exactly quantify the fluorescence induced by the exposure of a sample.

The fluorescence information received at the detector in the detection step is used in the evaluation step for the qualitative and / or quantitative detection of the investigated target molecules. They can be read out visually directly at the detector. Alternatively or additionally, a data processing system connected to the detector is used for forwarding, storing and / or evaluating it. In the course of the procedure any number of evaluation steps can be carried out. In particular, an evaluation step can be carried out as the last step of the method.

Embodiments of the method

According to an embodiment of the method, the sample providing step comprises applying the sample to be analyzed to the reaction area of the liquid receiving area and applying a protective liquid to the liquid receiving area.

Preferably, first the sample is applied to the reaction area and then the protective liquid is applied to the liquid receiving area surrounding the reaction area. The protective liquid is preferably a liquid which inhibits the evaporation of the sample to be analyzed, for example an oil-containing liquid. Advantageously, the protective liquid prevents or reduces vaporization of an underlying aqueous sample and / or reaction liquid.

According to one embodiment, the method comprises a detector temperature control step in which at least one detector of the optical waveguide device is temperature-controlled and the temperature of the detector is kept essentially constant.

The detector is preferably heated or cooled by means of a suitable tempering element at the beginning of the process by means of a Detektoremperemperierungseinrichtung to a predetermined temperature and maintained at about this temperature during the remaining process. A constant temperature of the detector has the advantage that detector-temperature-related measurement errors and / or measurement errors due to temperature drift effects in the detector can be avoided or reduced. Suitable detector temperatures are generally in the range of 15 ° C to 35 ° C. An adjustment of the detector temperature in the region of the room temperature is particularly preferred.

The Lichtleiterbündelfixierungsvorrichtung is tempered according to an embodiment of the method to a temperature between 50 ° C to 110 ° C, preferably between 60 ° C to 80 ° C, more preferably at 70 ° C.

Preferably, the Lichtleiterbündelfixierungsvorrichtung and the substrate temperature control are synchronously adjusted in time to approximately the same temperatures, so that the temperatures of the substrate and the sample-side Lichtleiterbündelenden permanently by less than about 20 ° C, preferably less than about 10 ° C, more preferably about 5 ° C different from each other. To simplify the control of the device, the optical fiber bundle ends may be adjusted to a constant temperature, which is preferably higher than the average temperature of the substrate. This temperature is preferably adjusted before the substrate is tempered by means of the substrate temperature control device.

In one embodiment of the method, the substrate tempering step comprises at least one tempering cycle with at least one heating and / or at least one cooling process.

Such Temperierungszyklen are particularly advantageous in certain procedures such as when performing a PCR. If a sample positioned on the substrate is to be subjected to a PCR, for example, the substrate may first be heated to 95 ° C., for example, and then tempered in several tempering cycles. Each tempering cycle includes, for example, a suitable primer annealing temperature, a suitable chain extension temperature, and a suitable denaturation temperature which, depending on the experiment, e.g. B. in the range of about 30 ° C to 70 ° C (Primeranlagerung) or about 50 ° C to 75 ° C (chain extension) or about 85 ° C to 100 ° C (denaturation) may be.

In each case, precisely one liquid receiving region or one reaction region contained in a liquid receiving region is exposed by exactly one light source by the optical waveguide device.

Furthermore, preferably, a plurality of liquid receiving regions can be exposed simultaneously by a light source and / or more than one light source can expose a liquid receiving region or its reaction region. In this way, for example, in the grid arranged on a substrate samples can be exposed line by line or column by column or in any other groups at the same time. Likewise, it is possible by means of such an optical waveguide device that light emitted by a plurality of light sources of different wavelengths is simultaneously directed to a single sample.

The fluorescence emanating from the sample to be analyzed in several liquid receiving areas or reaction areas can be detected, each with a separate detector. In one embodiment, the fluorescence of two or more liquid receiving areas or reaction areas is received or detected by the same detector. Embodiments are also possible in which light is received by exactly one liquid pickup area or reaction area of two or more detectors.

Computer program product according to one aspect

One aspect relates to a computer program product comprising computer-interpretable instructions stored on a computer-readable medium and / or as a computer readable data stream which, when loaded into the memory of a computer and executed by a computer, cause the computer to perform the method of the invention.

In order to control the method according to the invention, the computer-interpretable instructions or instructions are loaded into the memory of the optical waveguide device or a connected data processing system, for example a computer, and executed therein. A computer program product may be stored on a data carrier, e.g. As an optical disk, such as a CD, DVD, a magnetic data storage such. As a hard disk, a semiconductor memory, a magneto-optical memory, etc., or be provided as interpretable for a data processing system data stream.

The present invention is not limited to the above aspects and embodiments. Rather, individual features of one or more of the abovementioned aspects and / or embodiments or variants can be combined with one another to form further aspects and / or embodiments or variants of execution independently of the respective aspect and / or the respective embodiment or variant embodiment.

Description of the figures

An exemplary embodiment of the invention is described below with reference to exemplary figures, wherein

1 3 shows a side view of an optical waveguide device for emitting and receiving light with exemplary cross sections of the optical waveguide bundles,

2 shows a plan view of a liquid receiving area located on a surface,

3 FIG. 3 shows a side view of the exemplary system for exciting and measuring fluorescence on at least one reaction region, and FIG

4 a detail of the system 3 shows.

1 shows a schematic side view of the light guide device for emitting and receiving light. The optical waveguide device has at least one forked optical fiber bundle 10 put that into an excitation fiber bundle 12 and an emission fiber bundle 14 is divided. These forked fiber optic bundles 10 , may also be referred to as Y-fibers or Y-fibers, and may for example consist of individual glass fibers, or polymer-optical fibers, or a mixture of these. The one excitation fiber bundle end 16 of the excitation fiber bundle 12 is so in a light source side frame 18 fixed that the excitation fiber bundle end 16 one excitation light frequency filter each along the optical axis 20 , a light focusing element 22 , and a light source 24 , assigned. The excitation light frequency filter 20 , For example, may include a frequency filter and / or a band pass filter. The light focusing element 22 , For example, may be an optical lens of glass or transparent polymer or a innenverspiegelter hollow channel. The light source 24 For example, it may be a laser or a laser diode, an LED, an incandescent lamp, a gas discharge lamp. In one embodiment, not shown, for example, a light source with monochrome light can be used to avoid the use of excitation light frequency filters. The illustrated cross-section 26 the excitation fiber bundle end 16 together with the version 18 has a diameter of a few hundred microns to a few millimeters, for example from 100 microns to 5 millimeters, preferably from 500 microns to 3 millimeters, more preferably 2 millimeters

According to the illustrated embodiment, the other excitation optical fiber bundle end 28 of the excitation fiber bundle 12 together with the one emission fiber bundle end of the emission fiber bundle 14 so in a sample version 30 fixed that this version 30 in a Lichtleiterbündelfixierungsvorrichtung 32 can be arranged. In a further embodiment, the optical waveguide device may comprise two or more optical fiber bundles 10 include (not shown here), which in each case according to the above statements in a corresponding version 30 are fixed. Preferably, the Lichtleiterbündelfixierungsvorrichtung 32 made of a thermally conductive material, for example metal, preferably of aluminum or another light metal. In one embodiment, in the optical fiber fixation device 32 several holes provided in a given grid, which are just big enough to each one of the two or more versions 30 to be able to record. The version 30 and the optical fiber fixing device 32 but can also be made for example from one piece. Likewise, for example, the optical fiber without socket 30 directly in the optical fiber fixing device 32 be fixed. The illustrated cross-section 34 a sample side fiber optic bundle end 28 in the version 30 shows a homogeneous distribution of the individual light guides from one excitation and one emission light guide bundle 12 . 14 and has a diameter of a few hundred microns to a few millimeters, for example from 200 microns to 5 millimeters, preferably from 1 millimeter to 3 millimeters, more preferably 2.5 millimeters.

In the embodiment shown here are Lichtleiterbündelfixierungsvorrichtung 32 , as well as sample version 30 and the specimen-side optical fiber bundle end fixed therein 28 in thermal contact. The optical fiber fixing device 32 includes at least one sample-side heater 36 , preferably an electrically operated heating element, for. B. a Peltier element or a resistance heating element. However, other heating elements are also conceivable, for example based on pre-tempered fluids. By means of the sample-side heating elements 36 can the Lichtleiterbündelfixierungsvorrichtung 32 , any existing versions on the sample side 30 and the sample-side fiber bundle ends 28 be tempered.

In the detector-side emission optical fiber bundle end 38 are in the in 1 illustrated embodiment, all emission optical fiber bundles 14 the fiber optic bundle 10 the device all in a detector-side socket 40 fixed. This embodiment thus has only one detector-side emission optical fiber bundle end 38 on top of that, a detector 44 assigned. In the illustrated embodiment is also between the emission optical fiber bundle end 38 and the detector 44 an emission light frequency filter 42 arranged. As a detector 44 For example, a sensitive photodiode or a CCD, or CMOS sensor in question. The detector 44 can also be tempered in the embodiment shown here to avoid thermal noise in the measurement signal. This is a detector-side tempering 46 placed in close proximity to the detector to keep the detector at a constant temperature. The detector-side temperature control element 46 may be, for example, a Peltier element. In principle, the temperature control element 46 also from a combination of any heating element, for. B. a resistance heater, and any cooling element, for. B. a fan. The illustrated cross-section 48 the emission fiber bundle ends 38 in the detector-side version 40 has a diameter of a few millimeters to a few centimeters, for example from 1 millimeter to 2 centimeters, preferably from 5 millimeters to 1.5 centimeters, more preferably about 10 millimeters.

2 shows a plan view of one on a surface of a substrate 50 liquid intake area 52 , The substrate may be, for example, a glass or plastic slide. According to a preferred embodiment, on the substrate also a plurality of such liquid receiving areas 52 be arranged in an advantageous grid. Only one example of such a design of the substrate with 48 Liquid receiving areas represents the marketed by the company Beckman Coulter Biomedical GmbH (Munich, Germany) AmpliGrid.

The liquid intake area 52 comprises a central, substantially circular hydrophilic reaction region 54 whose diameter is about 1.6 mm in an advantageous embodiment. This reaction area 54 is surrounded by a substantially annular, hydrophobic central region 56 and an annular hydrophilic outer region surrounding this central region 58 , The this outer area 58 surrounding surface of the substrate 50 is essentially hydrophobic.

3 shows a schematic side view of the system according to the invention 60 for stimulating and measuring fluorescence. The system shown in one embodiment 60 shows an in 1 illustrated optical fiber device 62 for emitting and receiving light in conjunction with a substrate 50 , On the surface there is at least one liquid receiving area 52 , By means of at least one optical fiber bundle 10 , for example, with a sample-side version 30 the light guide bundle fixing device (not shown here in full) is fixed, can light on the liquid receiving area 52 and light is received from this. The substrate 50 is parallel to a substrate temperature control device in the embodiment shown here 64 positioned and is in direct thermal contact with it. The substrate tempering device 64 includes at least one heater 66 and at least one cooling device (not shown here). As an example of a substrate temperature control device, the AmpliSpeed marketed by Beckman Coulter Biomedical GmbH (Munich, Germany) may be cited. With such a temperature-controlled system 60 For example, a method according to the invention for the quantitative and / or qualitative detection of target molecules in a sample to be analyzed can be carried out.

4 shows a detail of the in 3 shown side view of the system 60 for stimulating and measuring fluorescence. According to one embodiment shows 4 the geometric arrangement of one in a sample-side version 30 fixed sample-side Lichtleiterbündelendes 28 relative to the reaction area 54 a liquid intake area 52 on a substrate 50 , As exemplified here, the version is 30 the optical fiber fixing device (not shown here) positioned so that the optical fiber bundle 10 with a liquid intake area 52 advantageously on the same optical axis 70 lies. The optical fiber bundle end surface 68 is also placed above a liquid receiving area so that it is at right angles to the optical axis 70 forms. This ensures that of the Lichtleiterbündelendfläche 68 outgoing excitation light on the liquid receiving area 52 meets. The light emitted from a fluorescent sample positioned in the liquid receiving region becomes the end surface of the optical fiber bundle 68 in the fiber optic bundle 10 added.

The physical nature of the individual optical fibers in the optical fiber bundle 10 define the Abstrahl- or acceptance angle α of the optical fiber bundle and thus according to generally known mathematical principles, the illuminated area. The distance a between Lichtleiterbündelendflächen 68 and the surface of the substrate 50 is thus advantageously chosen so that the illuminated area the reaction area 54 completely covers. The reaction area 54 has in the example shown here a diameter D, the Lichtleiterbündelendfläche 68 a diameter d. This results in a distance for the optical fiber bundle ends to the surface of the substrate of a = (D - d) / (2 · tan (α)).

Essentially, however, the distance a is greater than the total height of the liquid intake area 52 located liquid, that is, the overlaid with a hydrophobic liquid sample to avoid direct contact and contamination associated with the fiber optic bundle ends.

LIST OF REFERENCE NUMBERS

10

Light pipe

12

Excitation light guide bundle

14

Emission light guide bundle

16

Light source side excitation fiber bundle end

18

Light source side socket

20

Excitation light frequency filter

22

Light source side optical lens

24

light source

26

Cross section of an excitation fiber bundle end with socket

28

Sample side fiber optic bundle end

30

Sample-side version

32

Optical fiber cord fixation device

34

Cross section of a sample side fiber optic bundle end with socket

36

Heating device of the Lichtleiterbündelfixierungsvorrichtung

38

Detector side emission fiber bundle end

40

Detector-side socket

42

Emission light frequency filter

44

detector

46

Detector temperature control element

48

Cross section of the emission optical fiber bundle ends with socket

50

substratum

52

Liquid receiving area

54

reaction region

56

hydrophobic middle area

58

hydrophilic outer region

60

System for exciting and measuring fluorescence

62

Optical fiber device

64

Substrattemperierungsvorrichtung

66

Heating device of the substrate tempering device

68

Lichtleiterbündelendfläche

70

optical axis

Claims (20)

Optical waveguide device for emitting and receiving light, in particular for excitation and observation of fluorescence, having - an optical fiber bundle fixing device ( 32 ) and with At least one optical fiber bundle ( 10 ), wherein the at least one optical fiber bundle is fixed by means of Lichtleiterbündelfixierungsvorrichtung and is heatable by means of Lichtleiterbündelfixierungsvorrichtung. Optical fiber device according to claim 1, wherein the optical fiber fixing device ( 32 ) at least one heating device ( 36 ). Optical fiber device according to claim 1 or 2, wherein the optical fiber fixing device ( 32 ) at an end region of the at least one optical fiber bundle ( 10 ) is arranged. Optical fiber device according to at least one of the preceding claims, wherein the optical fiber fixing device ( 32 ) as a heating device ( 36 ) is trained. Optical fiber device according to at least one of the preceding claims, wherein the at least one optical fiber bundle ( 10 ) at least one excitation optical fiber bundle ( 12 ) with at least one excitation light guide and at least one emission light guide bundle ( 14 ) comprising at least one emission light guide. Optical fiber device according to claim 5, wherein the at least one optical fiber bundle ( 10 ) comprises more emission light guide than excitation light guide. Optical fiber device according to claim 5 or 6, having at least one light source ( 24 ) and at least one detector ( 44 ), wherein the at least one excitation optical fiber bundle ( 12 ) is associated with at least one light source and the at least one emission optical fiber bundle ( 14 ) is assigned at least one detector. Optical fiber device according to claim 7, wherein the detector ( 44 ) is temperature controlled. System for exciting and measuring fluorescence on at least one liquid pick-up area ( 52 ) with - a light guide device ( 62 ) according to one of the preceding claims, - a substrate ( 50 ), the surface of which comprises at least one liquid receiving area, and - a substrate tempering device ( 64 ), wherein the substrate is tempered by means of the substrate temperature control device and the optical waveguide device is arranged relative to the substrate, that the at least one optical fiber bundle ( 10 ) of the optical fiber device and the substrate are non-contact. A system according to claim 9, wherein said at least one optical fiber bundling device ( 32 ) fixed optical fiber bundles ( 10 ) above the substrate ( 50 ) and the shortest distance between the end face of the at least one optical fiber bundle and the substrate is between 0.1 mm and 20 mm, preferably between 0.5 mm and 10 mm, more preferably between 1 mm and 5 mm, and most preferably 3 mm , System according to claim 9 or 10, wherein the at least one liquid receiving area ( 52 ) a central, preferably circular hydrophilic reaction area ( 54 ), a preferably annular hydrophobic central region surrounding the reaction region ( 56 ) and a preferably annular hydrophilic outer region surrounding the middle region ( 58 ) and wherein the outer region is surrounded by a hydrophobic region. System according to at least one of claims 9 to 11, wherein at least one liquid receiving area ( 52 ) through the optical fiber fixing device ( 32 ) fixed optical fiber bundle end ( 28 ), the liquid receiving region and the optical fiber bundle end surface ( 68 ) of the associated optical fiber bundle ( 10 ) have substantially the same dimensions and the liquid receiving region and the associated optical fiber bundle are aligned with each other so that from the Lichtleiterbündelendfläche ( 68 ) Exiting light hits the assigned liquid receiving area. Method for qualitatively and / or quantitatively detecting target molecules in a sample to be analyzed, comprising: a sample preparation step, wherein a sample to be analyzed is located on at least one liquid receiving area ( 52 ) is provided on a substrate, - a substrate tempering step, wherein the substrate ( 50 ) by means of a substrate temperature control device ( 64 ), - a Lichtleiterbündeltemperierungsschritt, wherein at least one optical fiber ( 10 ) by means of a Lichtleiterbündelfixierungsvorrichtung ( 32 ) - an exposure step, wherein the at least one sample to be analyzed by means of a light guide device ( 62 an exposure step, wherein light coming from the sample to be analyzed is received by the light guide device, an evaluation step, wherein the light received in the detection step is used for qualitative and / or quantitative detection of the target molecules optionally present in the sample to be analyzed , The method of claim 13, wherein the sample providing step comprises applying a sample to be analyzed to the reaction region (12). 54 ) of the Liquid Reception Area ( 52 ) and applying a protective liquid to the liquid receiving area. Method according to claim 13 or 14, comprising a detector temperature control step, wherein at least one detector ( 44 ) of the light guide device ( 62 ) and the temperature of the detector is kept substantially constant. The method of claim 13 to 15, wherein the Lichtleiterbündelfixierungsvorrichtung ( 32 ) is tempered to a temperature between 50 ° C to 110 ° C, preferably between 60 ° C to 80 ° C, more preferably to 70 ° C. The method of claim 13 to 16, wherein the substrate tempering step comprises at least one Temperierungszyklus with at least one heating and / or at least one cooling process. The method of claim 13 to 17, wherein two or more reaction areas ( 54 ) simultaneously by a light source ( 24 ) and / or two or more light sources expose a reaction area. The method of claims 13 to 18, wherein light from two or more reaction areas ( 54 ) from the same detector ( 44 ) and / or receive light from exactly one reaction area of two or more detectors. A computer program product comprising computer-interpretable instructions which, when loaded into the memory of a computer and executed by a computer, cause the computer to perform that according to any one of claims 13 to 19.

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