WO2009019447A1

WO2009019447A1 - Illumination apparatus for use in fluorescence -based detection

Info

Publication number: WO2009019447A1
Application number: PCT/GB2008/002624
Authority: WO
Inventors: Alan Edward Green; Martin Alan Lee; David James Squirrell
Original assignee: Enigma Diagnostics Limited
Priority date: 2007-08-03
Filing date: 2008-08-01
Publication date: 2009-02-12
Also published as: GB0715168D0

Abstract

Epi-illumination apparatus for use in multiplexed fluorescence- based imaging or assays is provided with at least two different illumination sources and a beam split arrangement (130) for delivering radiation from at least a first of the sources to a sample location (105). Radiation from a second of the sources is provided with a transmission path that either bypasses the beam split arrangement or is delivered via a modified portion of the beam split arrangement. In an example, the beam split arrangement comprises a dichroic mirror (130) with a coated or embedded portion (200) providing a different material surface. This might for example either of fer. non-selective reflection of all incident radiation or wavelength-selective reflection which has a different characteristic from that of the dichroic mirror.

Description

ILLUMINATION APPARATUS FOR USE IN FLUORESCENCE-BASED DETECTION

The present invention relates to illumination apparatus for use in fluorescence-based detection methods and apparatus such as might be used in assays or imaging. It finds particular application in methods and apparatus for fluorescence microscopy and methods and apparatus based generally on fluorophore probes, including for example fluorescent resonant energy transfer.

In luminescence-based assay or imaging methods, susceptible molecules are excited by a stimulus, physical, chemical or mechanical, and subsequently emit light which can be detected. The emitted light can be used to find the position of the molecules and can thus be used in imaging, and/or to detect an involvement of the emitting molecule in a process.

A convenient form of stimulus is light which can be directed onto a sample containing the susceptible molecules and this form of luminescence is known as fluorescence. The emitted light has a wavelength or wavelength range different from that of the stimulating light so that it can be relatively easily detected in the presence of the stimulating light. Neither the stimulating light nor the emitted light is necessarily in the visible spectrum although where the emitted light is for instance in the near infra red it can be termed "non- fluorescent" or "dark" quenching and this is further mentioned below.

The materials providing susceptible molecules, or "fluorophores", are often referred to as dyes. An example of a fluorophore-based assay technique is fluorescent resonant energy transfer ("FRET") . Well known examples of dyes include fluorescein (blue excitation, green emission: usually used as a donor in FRET) and rhodamine (green excitation, red emission: often used as an acceptor in FRET) . Another example of a fluorophore-based assay technique is the use of dark quenchers which generally function using collisional or "Dexter" energy transfer rather than dipole-dipole as in FRET. The term "FET" (fluorescence energy transfer) covers both types as a generic term.

Using FET, the progress of a process in a sample can be determined from the emitted light. Susceptible moieties are present in two types, these types consisting of a donor moiety and an acceptor moiety. The moieties can take various forms such as separate fluorescent molecules or different fluorophore groups on the same molecule, or might not have a molecular form but be for instance particulate such as fluorescent beads or fluorescent quantum dots. On their own, donors fluoresce in response to stimulating light (also referred to herein as "excitation radiation", "illuminating radiation" or "stimulating light") delivered to the sample. However, if an acceptor is physically close by, there is a transfer of excitation energy between dipoles which will quench the donor's fluorescence and may instead lead to fluorescence by the acceptor. This is sometimes known as the "Forster" resonance energy transfer. The extent of energy transfer depends on the separation distance between the donor and acceptor. The FET assay techniques depend on the effect of a process on the physical proximity of donor/acceptor pairs. When they are sufficiently close together, there is transfer of energy from the donor to the acceptor and the overall fluorescent output of the pair is affected by that. When the pair is separated, the transfer of energy is reduced or stopped and the overall fluorescent output of the pair is detectably different. Thus a process which changes the physical proximity of donor/acceptor pairs in a sample causes a detectable change in the spectral content of light emitted by the sample, giving a measure of the progress of the process.

Susceptible fluorophores, including donor/acceptor pairs, are sometimes built into structures known as "probes". A probe has the character that it will behave in a certain way in a sample so that fluorescence emitted from the probe location will give useful information. For example, the probe might take up certain sites in which case the fluorescence will show those sites. In FET, the probe is often chosen so that it will be either constructed or divided by a process under assay, the donor and the acceptor being located on different parts of the probe so that they are brought spatially together or separated as the probe is constructed or divided. Probes may alternatively have more than one component, these components carrying a donor and an acceptor respectively and being either brought spatially close together or separated by the process under assay.

The change in physical proximity of a donor/acceptor pair can be detected in different ways. The acceptor may itself fluoresce when it receives energy from the donor, but at a different wavelength from that at which the donor fluoresces. Thus the change in physical proximity can be detected by measuring a change in the level of fluorescent output at the wavelength of either donor or acceptor. The rate of FRET is strongly dependent upon the donor-acceptor ("D-A") distance "r", being inversely proportional to r⁶. These distances are particularly convenient for studies of biological macromolecules as they are comparable to the diameters of many proteins, the thicknesses of biological membranes, and the distance between sites on multisubunit proteins. Any process that affects the D-A distance affects the energy transfer rate and allows the process to be quantified. For this reason FRET has been referred to as a spectroscopic ruler.

Whichever is monitored in order to detect that change is referred to as a "reporter", whether it is the donor or the acceptor. Some acceptors do not themselves fluoresce but merely "quench" the fluorescence of the donor. These (mentioned above) are known as "dark quenchers" and the donor in this case is necessarily the reporter. Monitoring or detecting arrangements incorporating a reporter can be generally referred to as reporter systems.

Acceptor molecules are also sometimes known as "receptors" but, depending on the process being monitored, there can be confusion with other elements of the process and the term acceptor is generally used herein.

FET assay techniques are used to monitor for example polymerase chain reaction ("PCR") processes and both FET and PCR techniques are discussed in ^"Real-Time PCR: An Essential Guide" edited by

Edwards, Logan and Saunders, published in 2004 by Horizon

Bioscience, ISBN 0-9545232-7-X. FET assay techniques may be used to monitor other types of nucleic acid amplification reactions, for example ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) , loop-mediated isothermal amplification

(LAMP) , rolling circle DNA amplification, multiplex ligation- dependent probe amplification (MLPA) and multiple displacement amplification.

It is known to multiplex measurements on one sample so that more than one process can be measured or monitored during the same time period. There may be more than one process that might occur in the sample and these may be separately detectable by using different dyes or probes. In FET, the probes themselves can be "labelled" by carrying different donor/acceptor pair combinations. For example, donor/acceptor pairs can differ in using different donor moieties, fluorescing at different wavelength ranges, or in using different acceptor moieties. If there is more than one type of donor, it is known to use a "universal acceptor" technique in which one type of acceptor accepts resonant energy from more than one type of donor. Depending on the types of donor, it may be necessary to provide excitation radiation of more than one wavelength. Similarly, if there is more than one type of acceptor, it is known to use a "universal donor" technique in which the resonant energy of one donor can be accepted by more than one acceptor. Although each probe type may carry the same donor, the different probe types are distinguishable by the fluorescence wavelength range of the respective acceptors. These techniques tend to be limited by the excitation range of the particular universal donor or acceptor dyes which can be used. For example, high excitation energies can be required by long wavelength dyes. Generally it is the fluorimeter design that limits multiplex analysis to either the universal donor or universal acceptor approaches. In systems optimised for the universal donor approach it is implicit that long wavelength dyes (intended for use as acceptors) may not be directly excited for application as donors. There is also a known method to get around this problem by using "big dye" Taqman® chemistries where a fluoroscein molecule is coupled to the reporter (donor) dye in an energy transfer pair. However, this significantly complicates the probe synthesis and associated costs. In systems configured for universal acceptor approaches, illuminating diodes are normally paired with specific detectors such that it may not be possible to apply the correct illumination required to carry out a multiplex universal donor experiment in an ideal excitation source/ emission detector combination or the system may lack a colour compensation system that is essential in order to deconvolute the overlapping spectra in a multiplexed universal donor experiment.

Optically-based strategies for achieving a flexible approach to multiplexing using the universal acceptor approach include the use of a high-powered excitation source, such as a gas cooled argon laser, and the use of multiple excitation source/fluorophore pairs, each source being matched to the peak excitation wavelength of its respective fluorophore . A high powered excitation source can direct sufficient energy to excite a number of different fluorophores directly, although multiple fluorophores can then be co-excited which requires that a colour compensation algorithm be used to deconvolute the mixed spectral output. A high power source can also have the disadvantage that universal donor systems do not work well since the acceptor (reporter) dyes can be already significantly excited by the source. The use of multiple excitation source/fluorophore pairs does offer multiplexing but requires more than one excitation source for illuminating a sample. Depending on the specific process involved, this can be physically difficult due to space limitations .

In another approach to multiplexing, non-optical techniques have also been used. For example, PCR products can be differentiated during amplification by plotting and analysing melting curves whose shape is a function of content, length and sequence of the product chains and this "melting peak analysis" can be used to distinguish different processes occurring in a sample, but these additional techniques are not available in all fluoroscent assay formats. For example, melting peak analysis cannot be used with a homogeneous format Taqman® (universal acceptor) arrangement since the probe produces a very poor melting motif.

There are different forms of illumination used in applying a stimulating light. In "transillumination", the stimulating light is delivered to one area of the surface of a sample and emitted light is collected from another area. For example, if the sample is held in a capillary tube, it can be illuminated from one side while emissions are detected on the other side of the capillary tube. However, in "epi-illumination", the emitted light may be collected from the same or an overlapping area of the surface of the sample as the area where the stimulating light is applied. This leaves considerably less space for excitation sources and detectors to be assembled. In at least some known arrangements for delivering stimulating light to and collecting emitted light from a sample, some optical components are shared and this finds particular application in epi-illumination apparatus. For example, stimulating light may be reflected to a sample by use of a dichroic mirror together with a lens . A dichroic mirror is usually characterised by the wavelengths that it will reflect, generally transmitting all the rest. The dichroic mirror thus provides filtering of the delivered stimulating light to lie within a known waveband or wavebands, according to its reflective characteristics, and the lens focuses the filtered light onto the sample. Light emitted from the sample and having a wavelength characteristic outside the known waveband or wavebands of the dichroic mirror is then picked up by the lens and delivered back to the dichroic mirror which transmits it to receiving equipment. Any light in the known waveband or wavebands will be reflected away from the receiving equipment by the dichroic mirror.

An overall arrangement for epi-illumination using a dichroic mirror might thus be that a stimulating light beam is shone onto the dichroic mirror at for instance 45°. The mirror reflects a filtered beam through a right angle onto the sample via a lens. Emitted light is picked up by the same lens and returned along the same path but in the other direction, again meeting the mirror at 45°. As long as the emitted light has a different wavelength from that at which the mirror will reflect it, the emitted light will now pass through the mirror and can be picked up by a detector placed behind the mirror with respect to the sample.

This general arrangement based on a mirror and light gathering device (the lens) suffers from the limitation imposed by the reflective characteristics of the mirror. Only stimulating light that will be reflected by the mirror and is a different wavelength from detectable emitted light can be applied to the sample and this in practice can limit the arrangement to only one stimulating wavelength or waveband. If the technique is based on a universal acceptor, then this limitation can mean only one process can be tracked in a sample at one time.

According to a first aspect of embodiments of the present invention, there is provided illumination apparatus for use in detection of fluorescence from a sample in an illuminated sample location, the apparatus comprising: a wavelength selective reflector for reflecting radiation having a selected wavelength characteristic; a light gathering device; and at least two transmission paths for directing illuminating radiation from at least two radiation source locations, in use of the apparatus, to the sample location, wherein at least a first of the transmission paths includes reflection at the selective reflector and at least a second of the transmission paths at least substantially excludes reflection at the selective reflector, the selective reflector being arranged to transmit fluorescence radiation not having the selected wavelength characteristic to a detector location, the fluorescence radiation having been gathered from the sample location by the light gathering device.

In arrangements according to this first aspect of the present invention, separate illuminating sources can be provided but only one detector may be necessary, picking up fluorescence radiation gathered by the light gathering device which is then transmitted via the selective reflector to the detector.

It may not be essential that the second of the transmission paths totally excludes reflection at the selective reflector but this may be the case Embodiments of the present invention can provide a particularly, spatially efficient arrangement for illuminating a sample with radiation from more than one different source while using a single detector for detecting fluorescence radiation which has been output by the sample in response to the more than one different source.

A known example of a selective reflector is a dichroic mirror. In order to provide the at least two transmission paths, it is possible to provide the first of the transmission paths so that it includes the mirror while the second of the transmission paths avoids, or "bypasses", the dichroic mirror but meets the sample location within the acceptance angle of the sample location for illuminating radiation.

Embodiments of the invention find particular application where the technique employed for illuminating a sample is epi- illumination since this a more spatially demanding arrangement than for example transillumination. Epi-illumination has the advantage that excitation radiation is delivered to the sample location in a direction away from the detector and will not therefore be detected thereby, in contrast to at least some transillumination arrangements.

Where the illumination apparatus is to be used in an epi- illumination configuration, it may still be physically difficult to provide the second of the transmission paths, bypassing the selective reflector, while still being able to site the detector so as to detect fluorescence radiation emitted from the sample location in response to illuminating radiation from the at least two different source locations. A solution to this is to use as the selective reflector a dichroic mirror in combination with a surface which is differently reflective. Both the first and second transmission paths may then be closely related, the first transmission path hitting the dichroic mirror and the second transmission path hitting the differently reflective surface. Illuminating radiation from both source locations can then follow parallel or nearly parallel paths.

The differently reflective surface might be provided within the general outline presented by the dichroic mirror to the source locations in use of the apparatus, for instance by coating or replacing a portion of a dichroic mirror. This provides a particularly space-efficient embodiment of the invention.

However, it might also be provided as an extension to said general outline, for instance as a protuberance, thus avoiding any loss of radiation reflected by the dichroic mirror.

Embodiments of the invention are not limited to just two transmission paths from excitation source locations to a sample location. For instance, a dichroic mirror might have more than one differently reflective area or a combination arrangement might be used in which a first excitation source is directed to the selectively reflective surface of the dichroic mirror, a second excitation source is directed to a coated or replaced portion of the dichroic mirror and a third excitation source is directed past the dichroic mirror assembly altogether.

According to a second aspect of the present invention, there is provided a dichroic mirror for use in illumination apparatus according to an embodiment of the present invention in its first aspect, the dichroic mirror comprising a selectively reflective surface in combination with an area having a differently reflective surface.

The differently reflective surface might comprise a different material, either as a coating or substituted for a portion of material of the dichroic mirror, for instance being embedded in it. For example, the differently reflective surface might be provided as a mirrored spot on the surface of an otherwise conventionally manufactured dichroic mirror. "Differently reflective" in this context is intended to mean having a different coefficient of reflection for at least one wavelength or wavelength band of incident radiation. For example, the differently reflective surface might reflect all incident wavelengths and thus be entirely non-selective, or it might select a wavelength or waveband for reflection different from that of the dichroic mirror. In the latter case, the differently reflective surface might indeed be provided by a secondary dichroic mirror, for instance by structuring material layers differently in or on a region of a primary dichroic mirror .

Embodiments of the present invention can offer flexibility and future proofing for various chemical fluorescence-based technologies in particular by:

• providing for universal acceptor and donor arrangements

• supporting multiplexing for example capable of monitoring at least four target processes for most relevant chemical technologies • low cost arrangement with as few excitation (or "illumination") sources as possible

• rapid optical collection of multiple channels simultaneously

• avoiding moving parts such as filter wheels, shutters and the like

This is done in embodiments of the present invention by offering an optical arrangement that combines a novel two source excitation strategy combined with a beam split optical arrangement. Known arrangements using beam split devices, such as a dichroic mirror, have been limited to one excitation source because known beam split devices have limited wavelength characteristics which only provide the necessary selective reflection for radiation from one source. Thus it has not been known to combine (and preferably bandwidth) two excitation sources in a beam split arrangement as it is counter-intuitive.

In one embodiment, the two or more illuminating sources are arranged to irradiate the sample with excitation radiation of different wavelengths over the same time period. A modulation arrangement is provided for modulating the excitation radiation of each of the at least two illuminating sources according to different respective modulation regimes and an emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the sample to said at least two sources can be separately detected. This modulation arrangement allows different measurements in one sample to be multiplexed. A known method and apparatus of modulating the excitation radiation of at least two illuminating sources and separately detecting the responses of the sample by the different modulation regimes is disclosed in international patent application WO 2007/066126, which is incorporated by reference.

One form of the solution according to an embodiment of the present invention is to use a mirrored spot on a dichroic mirror of otherwise known type in order to reflect radiation from a relatively longer wavelength source amongst multiple sources into a sample. Radiation from a relatively shorter wavelength source which is selectively reflected by the dichroic mirror need only lose a small percentage of signal due to the mirrored spot as long as the area of the spot is kept to a small value in relation to the surface area of the dichroic mirror.

A single light emitting diode ("LED") as an illumination source in an embodiment of the present invention can be tuned (by changing its drive power) to excite two or three fluorophores directly. A two-LED based system should therefore be able to excite at least four dyes directly. Therefore this fairly simple optical arrangement should be able to deliver a multiplex arrangement for tracking at least four processes, for instance in either a universal acceptor dye arrangement or a universal donor system.

Illumination apparatus suitable for use in fluorescence based assays will now be described as an embodiment of the present invention, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows a schematic representation in plan view of the apparatus;

Figure 2 shows in more detail a first delivery arrangement for delivering illumination radiation to a sample in use of the apparatus of Figure 1;

Figure 3 shows in more detail a second delivery arrangement for delivering illumination radiation to a sample in use of the apparatus of Figure 1;

Figure 4 shows a first modified dichroic mirror for use in the apparatus of Figure 1;

Figure 5 shows a second modified dichroic mirror for use in the apparatus of Figure 1; and

Figure 6 shows another alternative delivery arrangement using a dichroic mirror for use in the apparatus of Figure 1.

Referring to Figure 1, a sample for fluorescence-based assay is delivered in known manner, via a sample delivery input 100, to a

15mm glass capillary coated in an electrically conductive polymer ("ECP") to make a capillary assembly 105. The capillary assembly 105 is provided with a heating circuit 115 to deliver heat via the ECP and with an infrared thermopile 110 for dynamic feedback control to the heating circuit 115. Excitation radiation 170, 171 for use in exciting fluorescent probe activity is delivered to the capillary assembly 105 from two sources 145, 160, via respective lenses 140, 155, a half mirror

135, a dichroic mirror 130 and a further lens 125. The capillary itself has a beaded end 120 through which it receives the excitation radiation 170 and delivers fluorescent output. Such arrangements are of known general type.

The dichroic mirror 130 is adapted to reflect radiation at wavelengths of the excitation radiation, and thus delivers the excitation radiation via the further lens 125 to the capillary assembly 105, but to transmit radiation at the wavelengths of fluorescent probes present in the sample. Fluorescent radiation

175 emitted from the sample can thus be gathered by the further lens 125 and delivered along a different light path by the dichroic mirror 130 to a radiation detector 150.

Filters 195, 197 can be used at the output of each source 145, 160 to limit the excitation radiation 170 to desired wavelengths and to block parasitic excitation, although this may not be necessary where either of the sources 145, 160 is a laser since these can produce sufficiently narrow linewidths. A long pass filter 196 is used at the input to the detector 150 to reduce noise from sources other than the fluorescent probes present in the sample.

Referring to Figure 2, the dichroic mirror 130 itself is of known general type, being made from multiple dielectric layers (typically tens) of metal salts on a glass substrate. The particular metal salts are chosen according to their refractive index, which, in combination with the layer thicknesses, give the desired response. Dichroic mirrors are discussed for example in the "Encyclopedia of Laser Physics and Technology", published by RP Photonics Consulting GmbH.

The mirror 130, in use of the apparatus in a first arrangement, receives excitation radiation 170, 171 from two sources 145, 160. One of these sources 145 produces a beam 170 which is relatively broad in cross section, in the blue waveband of the electromagnetic spectrum, while a second of these sources 160 produces a beam 171 which is considerably narrower in cross section, in the red waveband of the electromagnetic spectrum. Both beams 170, 171 are reflected at the mirror 130 but in different ways. The reflective characteristic of the layered body of the mirror 130 selects the blue waveband for reflection and the beam 170 which is relatively broad in cross section is therefore reflected towards the sample present in the capillary assembly 105. The red waveband would not however be selected for reflection. The beam 171 which is considerably narrower in cross section, the red beam 171, is instead reflected by a mirrored spot 200 on the centre of the dichroic mirror 130.

The mirrored spot 200 has at least substantially the same cross section as the red beam 171 for maximum efficiency. If it is larger, fluorescent signal will be lost unnecessarily as the spot 200 blocks the fluorescent signal on its way to the detector 150. If it is smaller, some of the red beam 171 will miss the spot 200 and be wasted. The size of the red beam 171 itself is determined by the nature of its source. For example, this might be a laser in combination with a collimating lens (not shown) . The size of beam 171 produced by the collimating lens is a design choice but could be quite small, for example of the order of less than lmm.

The size of the cross section of the blue beam 170 is less critical. As long as the spot 200 is a broadband reflector, it will also reflect the blue beam 170 towards the sample present in the capillary assembly 105.

The mirrored spot 200 is a reflective coating which could be provided for example in a metal, such as silver, aluminium or gold, or itself could comprise a dielectric multi-layer mirror.

Dichroic mirrors 130 can be formed by known sequential vacuum deposition of the layers, typically by evaporation, onto a transparent substrate such as glass. The spot 200 could also be put on by vacuum deposition, which could be evaporation or sputtering. A contact mask would be used to define the area to be coated.

Concerning bandwidth characteristics of the two illuminating beams 170, 171, the red beam 171 only has to be narrow band from a fluorescence excitation standpoint. The source of the red beam itself doesn't have to be narrow band (although it will be if it is a laser) , as it can be narrow band filtered before it reaches the mirrored spot 200. The mirrored spot 200 itself can be made to be a narrow band reflector if it were a dielectric reflector although this is generally more problematic to make as it requires multiple layers in the same manner as dielectric beamsplitters .

Referring to Figure 3, in an alternative arrangement for embodiments of the present invention, the narrower of the two illumination beams 170, 171 is caused to "bypass" the dichroic mirror 130 in a different way. In this arrangement, the optical acceptance angle of the capillary assembly 105 with respect to illumination radiation is greater than the numerical aperture (acceptance angle) of the light gathering lens 125. In this case, there is an opportunity to angle the relatively narrow red beam 171 past the mirror 130 and onto the sample, outside the acceptance angle of the light gathering lens 125 but within the acceptance angle 300 of the capillary assembly 105. This avoids having to put a mirrored spot 200 onto the dichroic mirror 130.

Referring to Figure 4, a modified dichroic mirror 130 that might be used in embodiments of the present invention might incorporate a mirrored spot 200, or alternatively an embedded section having differently reflective properties, within the general outline presented by the mirror 130 to at least two source locations in use of the apparatus, such as the two sources 145, 160 shown in Figure 1. This arrangement of Figure 4 could be considered as providing a second reflective surface which is integral with that of the primary selectively reflecting surface, in this case the dichroic mirror 130, because the second reflective surface is carried on or in it and presents a reflecting profile which is continuous with that of the primary selectively reflecting surface .

Referring to Figure 5, an alternative modified dichroic mirror 130 to that of Figure 4 might incorporate a second reflective device 500 mounted on or adjacent to its periphery. This version allows for modification of existing dichroic mirrors.

Referring to Figure 6, where the selective reflector 130 is at least substantially transparent to the radiation produced by one of the illumination sources, it becomes possible to mount a second reflector 500 behind it, for instance sharing the same optical axis 600, instead of modifying the surface of the existing selective reflector 130. However, some radiation may be lost in transmission at the primary selective reflector 130.

A similar arrangement to that of Figure 6 would also be achieved by reversing the orientation of the mirror 130 of Figure 4 so that the second reflective surface 200 is on the back of the mirror 130 with respect to the illumination source (s) .

It will be understood that the embodiments of the invention described above and shown in the figures are examples of epi- illumination arrangements in which illumination is provided on the same side of the sample as detection (as opposed to transillumination) . Embodiments of the present invention are particularly useful in this case as a way of separating the illumination and fluorescent light, given that the particular wavelengths used will often prevent this being done by wavelength alone.

Claims

1. Illumination apparatus for use in detection of fluorescence from a sample in an illuminated sample location, the apparatus comprising: a wavelength selective reflector for reflecting radiation having a selected wavelength characteristic; a light gathering device; and at least two transmission paths for directing illuminating radiation from at least two source locations, in use of the apparatus, to the sample location, wherein at least a first of the transmission paths includes the wavelength selective reflector and at least a second of the transmission paths at least substantially excludes the wavelength selective reflector, the wavelength selective reflector being arranged to transmit fluorescence radiation not having the selected wavelength characteristic to a detector location, the fluorescence radiation having been gathered from the sample location by the light gathering device.

2. Apparatus according to Claim 1, further comprising two or more illuminating sources at said at least two different source locations .

3. Apparatus according to either one of the preceding claims wherein the wavelength selective reflector comprises a dichroic mirror.

4. Apparatus according to any one of the preceding claims adapted for use in epi-illumination of a sample.

5. Apparatus according to any one of the preceding claims comprising at least two illuminating sources and a detector, the detector being arranged to pick up fluorescence radiation gathered by the light gathering device which is then transmitted via the wavelength selective reflector to the detector.

6. Apparatus according to any one of the preceding claims wherein the wavelength selective reflector comprises a dichroic mirror.

7. Apparatus according to any one of the preceding claims wherein the second transmission path includes reflection at a reflective surface having one or more reflective properties different from one or more reflective properties of the wavelength selective reflector.

8. Apparatus according to Claim 7 wherein the reflective surface of the second transmission path is integral with the wavelength selective reflector.

9. Apparatus according to Claim 8 wherein the reflective surface of the second transmission path comprises a coated portion of the wavelength selective reflector.

10. Apparatus according to Claim 8 wherein the reflective surface of the second transmission path is provided by a substituted portion of the wavelength selective reflector.

11. Apparatus according to any one of Claims 8 to 10 wherein the differently reflective surface is provided within the general outline presented by the wavelength selective reflector to at least two source locations in use of the apparatus.

12. Apparatus according to any one of Claims 8 to 10 wherein the differently reflective surface is provided outside the general outline presented by the wavelength selective reflector to at least two source locations in use of the apparatus.

13. Apparatus according to any one of the preceding claims wherein the differently reflective surface is non-selectively reflecting .

14. Apparatus according to any one of Claims 7 to 13 wherein the differently reflective surface is positioned behind the wavelength selective reflector in use of the apparatus with respect to a source location of the first transmission path.

15. Apparatus according to any one of Claims 1 to 6 wherein the second of the transmission paths avoids the wavelength selective reflector but meets the sample location within the acceptance angle of the sample location for illuminating radiation.

16. Apparatus according to any preceding claim adapted to monitor a nucleic acid amplification reaction.

17. Apparatus according to claim 16 wherein the nucleic acid amplification reaction comprises a polymerase chain reaction.

18. A dichroic mirror for use in illumination apparatus according to any one of the preceding claims, the dichroic mirror comprising a selectively reflective surface in combination with at least one area having a differently reflective surface.

19. A dichroic mirror according to Claim 18 wherein the differently reflective surface is provided by a coating on the selectively reflective surface of the dichroic mirror.

20. A dichroic mirror according to Claim 18 wherein the differently reflective surface is provided by a material embedded in the selectively reflective surface of the dichroic mirror.

21. A dichroic mirror according to any one of Claims 18 to 20 wherein the differently reflective surface is provided as a mirrored spot on the surface of the dichroic mirror.

22. A dichroic mirror according to any one of Claims 18 to 20 wherein the differently reflective surface is provided by a further dichroic mirror.

23. Apparatus according to any of claims 1-17 further comprising two or more illuminating sources at said at least two different source locations, wherein said at least two sources irradiate the sample with excitation radiation of different wavelengths over the same time period, a modulation arrangement for modulating the excitation radiation of each of said at least two illuminating sources according to different respective modulation regimes and wherein an emission detector is adapted to detect each of said different respective modulation regimes in emitted radiation such that responses of the sample to said at least two sources can be separately detected.