GB2500177A

GB2500177A - Fluorometer with beamsplitter

Info

Publication number: GB2500177A
Application number: GB1204063.0A
Authority: GB
Inventors: James Morris; Julian Nicholson; Matthew Quartley
Original assignee: Valeport Ltd
Current assignee: Valeport Ltd
Priority date: 2012-03-07
Filing date: 2012-03-07
Publication date: 2013-09-18
Also published as: GB201204063D0

Abstract

A monostatic fluorometer comprises light source 30 to transmit an excitation beam 110; detector 60 to detect fluorescent emissions 130; and beamsplitter 90 to direct the excitation beam over an excitation path 120 into a fluorescent target 100 to generate emissions; the beamsplitter receiving the emissions over an emission path defined by a numerical aperture of the detector 60 and the beamsplitter 90 which is parallel to the excitation path, the emission beam passing through the beamsplitter and onto the detector. The beamsplitter enables the excitation beam to be coaxially aligned with an emission beam received by a detector which increases overlap volume of the device and enables measurements at distances closer to the device than possible with a multistatic device which increases sensitivity. The fluorometer can be made more compact than is possible with a multistatic device and can be air-free which makes it suitable for submersible applications.

Description

FLUOROMETER

FIELD OF THE INVENTION

The present invention relates to a fluorometer and in particular an assembly for a 5 fluorometer.

BACKGROUND

Fluorometers are known. Typically, a fluorometer is used to determine the fluorescent properties of certain compounds present in a material such as, for example, a fluid 10 which enables the concentration of the compound to be determined. This is particularly useful in the aquatic environment where concentrations of, for example, chlorophyll, rhodamine, crude or refined oils, fluorescein and others may be determined.

15 Although existing fluorometers enable measurement of some compounds, their performance can still be limited. Accordingly, it is desired to provide an improved fluorometer.

SUMMARY

20 According to a first aspect, there is provided a monostotic fluorometer or monostotic fluorometer assembly, comprising: a light source operable to transmit an excitation beam; a detector operable to detect emissions or an emission beam; and a beam splitter operable to direct the excitation beam over an excitation path into a fluorescent target to generate the emissions or emission beam in response to the 25 excitation beam, the beam splitter being operable to receive the emissions or emission beam over an emission path. The emission path may be defined by a numerical aperture of the detector and beam splitter assembly or arrangement. The emission path may be coaxial with or parallel to the excitation path. The emissions or emission beam may then pass through the beam splitter and onto the detector.

30

The first aspect recognises that a problem with existing fluorometers is that their sensitivity can be poor and their size is larger than desirable. In particular, existing fluorometers utilise a multi-static arrangement where an excitation beam is transmitted into a target from a light source and the emission beam is detected by a detector 35 which is spatially offset from the light source. In such a multi-static arrangement, both the light source and the detector are orientated to face towards the target; this causes the size and sensitivity of the device to be limited by the geometry of this arrangement. In particular, it is typically necessary to orientate both the light source and the detector

1

towards the target in order to minimise scattering due to turbidity and to minimise any cross-talk or cross-coupling. However, such an arrangement limits the minimum size of the device. Also, the sensitivity of the device is limited by the volume of the target for which the emission beam and the field of view of the detector intersect as well as the 5 distance from the device to the intersection point.

Accordingly, a fluorometer or fluorometer assembly is provided. The fluorometer or fluorometer assembly may be a monostotic fluorometer or fluorometer assembly. The fluorometer or fluorometer assembly may comprise a light source which transmits an 10 excitation beam. The light source may transmit the excitation beam in response to a voltage or current applied to the light source. A detector may be provided which detects emissions or an emission beam. The detector may output a voltage or current in response to detected emissions. A beam splitter may be provided which directs the excitation beam into a fluorescent target. The fluorescent target may then generate 15 emissions or an emission beam in response to the excitation beam. The beam splitter may then receive the emissions or emission beam. The beam splitter may receive the emissions or emission beam over an emission path defined by a numerical aperture of the detector and the beam splitter assembly or arrangement which is coaxial with or parallel to the excitation path. The emission beam may then pass through the beam 20 splitter and be detected by the detector.

Utilising a beam splitter provides for many advantages. First, the beam splitter enables an excitation beam to be parallel to or coaxially aligned with an emission beam received by a detector. Aligning the excitation beam with the emission beam 25 significantly increases the beam overlap volume of the device since the intersection between these beams is greater than is possible with offset beams such as those provided by multi-static or multi-aperture arrangements. Hence, unlike in those multi-aperture arrangements where the excitation beam and the detected emission beam pass through different apertures or windows, the first aspect may provide a mono-30 aperture arrangement where both the excitation beam and the detected emission • beam pass through the same aperture or window. Increasing the beam overlap

; volume of the fluorometer increases its sensitivity. Also, by arranging for the excitation

• •

beam and the emission beam to be aligned enables measurements to be made at distances which are significantly closer to the device than would be possible with a 35 multi-static device where the minimum distance is dictated by the relative angle of intersection between the excitation beam and the emission beam. Making

• • •

measurements at distances which are closer to the device again increases sensitivity.

• I. Secondly, using a beam splitter to redirect the excitation beam reduces the

geometrical constraints on the placement of the light source with respect to the detector which enables the light source to be placed closer to the detector and even orientated towards the detector without causing an increase in any cross coupling. Hence, the fluorometer can be provided in a much more compact arrangement than 5 is possible with a multi-static or multi-aperture device. Also, it is possible to provide an air-free device which makes it particularly suitable for submersible applications.

In one embodiment, the light source transmits the excitation beam to the beam splitter over a transmission path and the emissions or emission beam passes through the beam 10 splitter to the detector over a reception path, the transmission path and the reception path being non-parallel or non-coaxial. Having transmission paths and reception paths which are not parallel or not coaxial enables a more compact arrangement to be provided.

15 In one embodiment, the transmission path and the reception path are generally orthogonal. Accordingly, a degree of orthogonality may be provided between the transmission path and the reception path. That is to say, the two paths need not necessarily be perpendicular, just that their relative orientations have an orthogonal component to provide for a non-parallel angular relationship between the two paths.

20

In one embodiment, the beam splitter is transparent to the emissions or emission beam and the reception path is parallel to or coaxial with the excitation path and the emission path. Accordingly, the reception path may also be parallel to or coaxially aligned with both the excitation path and the emission path.

25

In one embodiment, the beam splitter comprises a partially reflective mirror. It will be appreciated that a partially reflective mirror provides for a simple and effective beam splitter.

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30 In one embodiment, the beam splitter comprises a dichroic mirror.

In one embodiment, the dichroic mirror is transparent to the emissions or emission beam and the detector is aligned to detect the emissions or emission beam over the emission path. It will be appreciated that it is possible to select properties of the dichroic mirror 35 which are able to change the path of the excitation beam whilst not affecting the path of the emissions or emission beam. In these circumstances, the detector is positioned to detect the emissions or emission beam over the emission path, the emission path aligning with the reception path.

3

In one embodiment, the fluorometer comprises an absorber positioned so that the beam splitter is located between the absorber and the light source. Providing an absorber helps reduce the effects of any reflections within the fluorometer or in the 5 target volume. This helps to reduce cross-coupling and noise within the fluorometer.

In one embodiment, the absorber is operable to reduce reflections of any of the excitation beam not directed along the excitation path by the beam splitter which is received by the absorber. Providing an absorber helps reduce any reflections of any 10 light from the excitation beam which passes through the beam splitter which might otherwise be detected by the detector. This helps to reduce cross-coupling and noise within the fluorometer.

In one embodiment, spatial arrangement of the detector and the beam splitter defines 15 a void which is at least partially filled by a material which provides greater attenuation at frequencies other than a frequency of the emissions or emission beam. Accordingly, a material may be provided which attenuates frequencies other than the frequency of the emissions or emission beam in order to improve the signal to noise ratio of the fluorometer. The void may be air-free.

20

In one embodiment, the fluorometer comprises a light source filter operable to narrow a bandwidth of the excitation beam. Narrowing the bandwidth of the excitation beam helps to provide a more precise control over the frequency of the excitation beam and helps to provide for improved frequency discrimination between the excitation beam 25 and any emission beam. Again, this helps to improve the sensitivity of the device.

In one embodiment, the light source filter is positioned between the light source and the beam splitter.

30 In one embodiment, the fluorometer comprises a detection filter operable to provide greater attenuation at frequencies other than a frequency of the emissions or emission beam. Providing a detection filter narrows the frequency band of light received by the detector, which again helps to improve the signal to noise ratio and improves the sensitivity of the device.

35

In one embodiment, the detection filter is positioned between the beam splitter and the detector.

• • i

4

In one embodiment, the fluorometer comprises a pair of adjacent prisms positioned to orientate the beam splitter. Providing an adjacent pair of prisms helps to define the geometry of the optical components and provides for a robust and reliable arrangement which is simple to manufacture.

5

In one embodiment, hypotenuse surfaces of the pair of prisms are positioned adjacent to each other to orientate the beam splitter. Accordingly, the beam splitter may be positioned along the hypotenuse of the prisms in order to hold the beam splitter in place and define the geometric relationship between the beam splitter, the detector, 10 the light source and the target.

In one embodiment, the detector is positioned on a cathetus surface of one of the pair of prisms with the light source is positioned on a cathetus surface of another of the pair of prisms. Accordingly, detector and light source may be positioned along surfaces of 15 the prisms in order to hold the detector and light source in place and define the geometric relationship between the beam splitter, the detector, the light source and the target.

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In one embodiment, the one of the pair of prisms provides greater attenuation at 20 frequencies other than a frequency of the emissions or emission beam. By providing a prism which attenuates light with greater effectiveness at frequencies other than the frequency of the emissions or emission beam, those other frequencies can be reduced, thereby improving the signal to noise ratio and sensitivity of the device.

25 In one embodiment, the fluorometer comprises a plurality of light sources, each being operable to transmit an excitation beam with a different frequency. Accordingly, different light sources may be provided which provides different frequencies in order to cause fluorescence of different substances or differential excitation of the same substance to allow fingerprinting according to absorption. For example, many algae 30 fluoresce at 685nm, but have very different absorption characteristics. By providing a plurality of such light sources, a single device can seek to measure multiple characteristics.

In one embodiment, the fluorometer comprises a plurality of detectors, each being 35 operable to detect emissions or an emission beam with a different frequency. By providing a plurality of detectors, again a number of different characteristics may be

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In one embodiment, the beam splitter is operable to direct the plurality of excitation beams into the fluorescent target to generate the plurality of emissions or emission beams and to receive the plurality of emissions or emission beams, the plurality of emissions or emission beams passing through the beam splitter onto the plurality of 5 detectors.

In one embodiment, the light source, the detector and the beam splitter together comprise a detection module, the monostatic fluorometer comprising a plurality of the modules. Accordingly, an assembly of the light source, the detector and the beam 10 splitter may together form a module and a fluorometer may comprise a plurality of those modules. It will be appreciated that the light sources and detectors of each module may emit and detect on different frequencies. Again this enables a single device to measure multiple characteristics.

15 In one embodiment, the fluorometer comprises a fluorescent layer operable to interact with the target, the fluorescent layer having a fluorescence which is modified by properties of the target. Accordingly, rather than detecting the direct fluorescent properties of the target itself, the fluorescent properties of a fluorescent layer may instead be detected, with those fluorescent properties changing dependent on 20 characteristics of the target. Hence, it can be seen that it is possible to measure characteristics of the target indirectly.

In one embodiment, the properties comprise chemical or physical properties of the target.

25

In one embodiment, the properties include at least one of a temperature of the target, a pH of the target, an amount of dissolved oxygen in the target and an amount of dissolved carbon dioxide in the target.

30 In one embodiment, the fluorescent layer is positioned in the excitation path and the layer generates the emissions or emission beam.

In one embodiment, the fluorescent layer is positioned adjacent to one of the pair of prisms. Accordingly, this arrangement enables the fluorescent layer to be positioned 35 adjacent to the prism which provides for a more compact arrangement than that of a multi-static fluorometer which would need the fluorescent layer to be positioned a minimum distance away from the device dictated by the geometry of the light source

6

and the detector. In one embodiment, the fluorescent layer is bonded to one of the pair of prisms.

In one embodiment, a frequency of the excitation beam is greater than a frequency of 5 the emissions or emission beam.

According to a second aspect, there is provided a method, comprising the steps of: transmitting an excitation beam; detecting emissions or an emission beam; and directing the excitation beam over an excitation path into a fluorescent target to 10 generate the emissions or emission beam in response to the excitation beam, receiving the emissions or emission beam over an emission path defined by a numerical aperture of a detector and a beam splitter assembly or arrangement which is coaxial with or parallel to the excitation path to be detected.

15 In embodiments, method steps are provided corresponding to embodiments of the first aspect.

According to a third aspect, there is provided a monostotic fluorometer or monostotic fluorometer assembly, comprising: a common aperture providing an excitation beam 20 from a light source over an excitation path into a fluorescent target and receiving emissions from said fluorescent target in response to said excitation beam; and an optical assembly operable to receive said emissions within a region defined by an acceptance angle of a detector and a beam splitter assembly or arrangement and to provide said excitation beam from a light source, said excitation beam and said region 25 being axially aligned.

In embodiments, features of the first aspect are provided as set out above.

Further particular and preferred aspects of the present invention are set out in the 30 accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

Where an apparatus feature is described as being operable to provide a function, it will 35 be appreciated that this includes an apparatus feature which provides that function or which is adapted or configured to provide that function.

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BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described further, by way of example only, with reference to preferred embodiments thereof as illustrated in the accompanying drawings, in which:

Figure 1 illustrates a combined prism and filter assembly according to one embodiment; Figure 2 illustrates a combined prism and filter assembly incorporating multiple light sources and detectors according to one embodiment;

Figure 3 illustrates a multiple prism and filter assemblies according to one embodiment; and

Figure 4 illustrates an oceanographic fluorometer according to one embodiment.

DESCRIPTION OF THE EMBODIMENTS Overview

Before discussing embodiments in any detail, first an overview will be provided. Embodiments provide an assembly which can be incorporated into an optical device such as, for example, a fluorometer which is used to measure the fluorescent properties of materials from which characteristics of those materials can be derived. For example, a fluorometer may be used to determine the presence and concentration of fluorescent substances within a fluid.

The assembly utilises a light source and a detector in combination with a beam splitter. The light source emits an excitation beam of light at a particular frequency which is directed by the beam splitter through a common aperture into a target. The excitation beam will typically illuminate a conical volume having an excitation axis. The target may contain material which emits light at another wavelength in response to the incident light (such as a fluorophore).

Although the emitted fluorescent light will generally be radiated omnidirectionally, a spherical sector of that omnidirectional emission will fall within the numerical aperture of the beam splitter/detector assembly or arrangement. This spherical sector may be considered to be analogous to an emission beam from the target. The numerical aperture will typically be defined by a conical volume having an emission axis.

The emission beam received through the common aperture by the beam splitter from the target is typically axially aligned with the excitation beam provided by the beam splitter to the target. Typically, the excitation axis and the emission axis are parallel. In symmetric arrangement, the excitation axis and the emission axis are coaxial. Hence, the assembly has a monostotic arrangement. The emission beam passes through the

beam splitter and is received by a detector. The detector may then determine the amplitude of the emission beam from which characteristics of the target may be derived.

5 Such an arrangement where the excitation beam and emission beam within the field of view of the beam splitter and detector assembly or arrangement are aligned in a coaxial, monostotic or monoaperture arrangement increases the overlap of the excitation and emission beams and reduces the distance from the target volume to the fluorometer window compared to that of a bi-static or multi-static arrangement where 10 only intersecting portions of the excitation beam and the field of view of the detector contribute to the measurement volume. This provides for a more compact assembly having greater sensitivity due increased beam overlap volume.

Combined Prism and Filter Assembly 15 Figure 1 is a schematic illustration of a combined prism and filter assembly, generally 10, according to one embodiment. In overview, this arrangement provides a pair of prisms 20, 50 between which is located a beam splitter 90. It will be appreciated that the beam splitter 90 may be provided as a coating on a substrate bonded between the pair of prisms 20, 50, or a coating on one or both of the pair of prisms 20, 50 and that 20 the pair of prisms 20, 50 may then be bonded together. A light source 30 is provided adjacent a first prism 20 and a detector 60 is provided adjacent a second prism 50. Light from the light source 30 travels first along the path 110 before being directed by the beam splitter 90 along the path 120 into a target 100. Light emitted by the target 100 in response is received over the path 130 by the beam splitter 90 and passes along 25 the path 140 to the detector 60.

Prisms

In this example, the first prism 20 has an isosceles right-angled triangle cross-section. However, it will be appreciated that other prism configurations are possible. Along a 30 surface 22 defined by a first cathetus of the right-angled isosceles triangle is disposed the light source 30 such as, for example, a light emitting diode or laser diode.

Positioned between the light source 30 and the first prism 20 is a light source filter 40. The light source filter 40 narrows the bandwidth of the light emitted by the light source 35 30. It will be appreciated that the light source filter 40 may be provided as a separate filter sandwiched between the light source 30 and the first prism 20, or may be a coating on either the first prism 20 or the light source 30.

9

The second prism 50 also has a right-angled isosceles triangle cross-section. The second prism 50 has a detector 60 positioned along a surface 52 of the second prism 50 defined by a first cathetus of the right-angled isosceles triangle.

5 Positioned between the second prism 50 and the detector 60 is a detection filter 70 which attenuates incident light at frequencies other than that intended to be detected by the detector 60. It will be appreciated that the detection filter 70 may be provided as a separate filter sandwiched between the detector 60 and the second prism 50, or may be a coating on either the second prism 50 or the detector 60.

10

Positioned along a surface 54 of the second prism 50 defined by a second cathetus of the right-angled isosceles triangle is a non-reflective layer 80. The non-reflective layer 80 acts to reduce any excitation light reflected from the surface 54 of the second prism 50. It will be appreciated that the non-reflective layer 80 may be a separate layer 15 abutting the surface 54 of the second prism 50, or may be deposited onto that surface 54.

The first prism 20 and the second prism 50 are positioned adjacent each other along their surfaces 26, 56 which are defined by the hypotenuse of their right-angled isosceles 20 triangles. Hence, the resultant prism structure resembles a cuboid.

Beam Splitter

Sandwiched between the first prism 20 and the second prism 50 along the surfaces 26, 56 defined by the hypotenuse of their respective right-angled isosceles triangles is a 25 beam splitter 90. The beam splitter 90 functions to interact with light passing through the two prisms 20, 50, as will be explained in more detail below. In this example, the beam splitter 90 is a dichroic mirror. It will be appreciated that the beam splitter 90 may be provided as a separate structure sandwiched between the first prism 20 and the second prism 50, or may be provided as a coating on either or both of the surfaces 26, 30 56 defined by the hypotenuse of their right-angled isosceles triangles.

Operation

The light source 30 emits an excitation beam. The excitation beam passes through the light source filter 40 to narrow the bandwidth of the excitation beam to a selected 35 frequency fl. The excitation beam travels through the first prism 20 over the path 110 until it is received by the beam splitter 90.

* • • • • « • • *

10

The optical characteristics of the beam splitter 90 are such that the excitation beam received over the path 110 onto the beam splitter 90 is mostly reflected along the path 120 through the first prism 20 and towards the target 100. Typically, the excitation beam will define a first conical volume defined by the numerical aperture of the combined 5 light source 30 and beam splitter 90 optical assembly or arrangement. The first conical volume extends away from the light source 30 over the path 110 towards the beam splitter 90 and then over the path 120 towards the target 100. In this arrangement, the excitation beam is generally symmetrical. However, it will be appreciated that non-symmetric beams may be formed.

10

The excitation beam interacts with the target 100. Substances within the target 100, such as fluorophores, respond to the excitation beam and fluoresce, generally at a specific frequency f2 or number of frequencies dependent upon the characteristics of the fluorophore. Those emissions from the fluorophores generally have a frequency 15 which is lower than that of the excitation beam.

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As shown in Figure 1, the emissions are generally omnidirectional, but a spherical sector within the field of view of the beam splitter 90 /detector 60 assembly or arrangement which is referred herein as an emission beam travels along the path 130. In other words, 20 the combined beam splitter 90 and detector 60 optical assembly or arrangement provide a second conical volume defined by the numerical aperture of the combined light source 30 and beam splitter 90 optical arrangement. The second conical volume extends away from the detector 60 over the path 140 towards the beam splitter 90 and then over the path 130 towards the target 100. In this arrangement, the conical volume 25 is generally symmetrical. However, it will be appreciated that non-symmetric conical volumes may be formed. In this example, the path 130 is coaxial with and opposite to the path 120. Hence, the excitation beam and emission beam can be considered to be coaxial. Accordingly, the first and second conical volumes are coaxially aligned and overlap on the target side of the beam splitter 90. However, other arrangements 30 are possible where the first and second conical volumes are simply parallel and overlap on the target side of the beam splitter 90

The emission beam passes through the first prism 20 and is received by the beam splitter 90. In this example, the characteristics of the beam splitter 90 are selected to allow the 35 emission beam to pass through the beam splitter 90 and continue along the path 140 through the second prism 50 until received by the detector 60.

11

Hence, it can be seen that this arrangement enables the excitation beam to be generated by the light source 30 out of the field of view of the detector 60, but then directed by the beam splitter 90 along the field of view of the detector 60 into the target 100. Also, the coaxial emission beam within the field of view of the detector 60 is 5 largely unaffected by the prisms 20,50 or the beam splitter 90 to enable the emission beam to be detected by the detector 60.

Such an arrangement provides for a particularly compact assembly 10 which achieves high sensitivity, is simple to manufacture and assemble, is robust, and can be 10 miniaturised. The assembly 10 is highly reliable and can be used in particularly harsh environments, such as being incorporated into oceanographic instruments. In particular, because the assembly 10 can have no air voids, it is not compromised by a deep-ocean, high pressure environment. Providing the excitation beam coaxially with the field of view of the detector 60 means that measurements can be made right at 15 the surface 142 of the first prism 20, rather than only being possible at a minimum distance from the assembly, such as would occur with a multi-static arrangement since no detection would be possible in a region closer than the point of intersection of the excitation beam and the field of view of the detector. By enabling the measurements to occur closer to the assembly 10, the sensitivity of the instrument is increased since the 20 amplitude of the emission beam reduces in proportion to the distance of the target volume from the detector 60. Accordingly, the assembly 10 can be surface mounted or even arranged to present its detection surface 142 effectively as a window on the outer surface of an instrument.

25 Also, as will be explained in more detail below, such an arrangement enables an embodiment which utilises an optional modifiable coating layer 155 along the surface 142 of the first prism 20.

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Noise reduction

30 The arrangement shown in Figure 1 incorporates a number of noise reduction techniques which further help to improve the sensitivity of the assembly 10.

As mentioned above, the provision of the source filter 40 helps to narrow the frequency band of the excitation beam in order to provide improved frequency discrimination 35 between the excitation beam frequency fl and the emission beam frequency f2.

The beam splitter 90 transmits approximately 80% of the excitation beam along the path 120, whereas around 20% of the excitation beam continues along the path 150

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into the second prism 50. However, it will be appreciated that the proportion light redirected can selected to vary typically between 50% and 99.99% and most likely the split will be 95%/5% to 98%/2%. The second prism 50 is made from a material which attenuates frequencies other than the frequency f2 of the expected emission beam. 5 Accordingly, by the time the 20% portion of the excitation beam which enters the second prism 50 reaches the non-reflective layer 80 it has been significantly attenuated.

On reaching the non-reflective layer 80, the excitation beam is further attenuated by 10 this non-reflective layer 80.

Any of the excitation beam which is reflected by the non-reflective layer 80 along a path opposite to the path 150 must again pass through and be attenuated by the second prism 50 prior to reaching the beam splitter 90.

15

Around 20% of the light that reaches the beam splitter 90 will pass back towards the light source 30 and 80% of the light that reaches the beam splitter 90 is reflected towards the detector 60. However, it will be appreciated that the proportion light redirected can selected to vary typically between 50% and 99.99% and most likely the 20 split will be 95%/5% to 98%/2%. However, any light which is left must again pass through the second prism 50 along the path 143 and is further attenuated.

Should any of the excitation beam reach the detection filter 70, then this remaining light is reduced or attenuated by the detection filter which attenuates frequencies 25 other than the frequency f2 of the expected emission beam.

Accordingly, it can be seen that as a result of this effective attenuation regime the light source 30 can be in close proximity to the detector 60 and even illuminate within the field of view of the detector 60 without causing any significant cross-coupling. Again, 30 this enables a robust and compact arrangement which can be miniaturised and which has high sensitivity.

Modifiable Coating Laver

Rather than using the assembly to directly illuminate a target 100, a modifiable coating 35 layer 155 may be optionally provided adjacent the surface 142 of the prism 20. The modifiable coating layer 155 may be optionally bonded to the surface 142 of the prism 20. The fluorescent properties of such a modifiable coating layer 155 are configured to change in response to characteristics of the target 100. Hence, such a modifiable

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coating layer 155 may be used to derive characteristics of the target 100 which would not normally be derivable by direct fluorescent measurements. This modifiable surface layer interacts with the target substance 100 and its fluorescent properties are changed in response to characteristics of the target 100.

5

For example, a modifiable coating layer 155 may be provided whose fluorescence changes based on the amount or concentration of dissolved oxygen or carbon dioxide in the target 100, or based on the temperature or pH of the target 100. It is then possible to, for example, determine the concentration of oxygen in the target based on 10 the fluorescent response of the modifiable coating layer 155.

It will be appreciated that many different modifiable coating layers may be provided which can provide an indication of many different characteristics of the target 100. Hence, the provision of such a modifiable coating layer 155 enables other 15 characteristics of the target 100 to be measured.

Multiple Light Sources and Detectors

It will be appreciated that rather than providing just one light source 30 and one detector 60, multiple light sources 30A - 30D and multiple detectors 60A - 60D may be 20 provided by the assembly 10E, as shown in Figure 2. Typically, each of the light sources 30A - 30D and detectors 60A - 60D may be optimised to measure different characteristics of the target 100. Each light source may be paired with a corresponding detector. Each light source and detector pair may also have associated filters and other noise reduction measures appropriate to improving their 25 sensitivity. Also, one or more light source and detector pair may be associated with a modifiable coating layer. This provides for a compact assembly which is able to measure many different characteristics of a target.

Figure 3 illustrates schematically an alternative arrangement showing multiple 30 assemblies 1 OA - 10D co-located together (and typically bonded together by bonding adhesive 300), where each assembly is provided with one or more different light sources and/or detectors. Also one or more light source and detector pair may be associated with a modifiable coating layer. Again, this enables the resultant assembly to detect multiple characteristics of the target 100 in a compact arrangement.

35

Figure 4 illustrates in more detail an example oceanographic fluorometer according to one embodiment, which incorporates multiple assemblies 10 extending circumferentially around an axial end of the fluorometer 200. These assemblies may

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comprise any of the assemblies 10, 10A - 10E mentioned above. Of course, it will be appreciated that the assemblies 10 could be provided on a circumferential surface 220 of the fluorometer 200.

5 Hence it can be seen that in embodiments, the detector field of view through the beam splitter defines the emission beam, and the emission beam and excitation beam are coaxial. In other words, the arrangement of the beam splitter enables a light source which emits an excitation beam which is non-axial with respect to the field of view of the detector to direct the excitation beam along the field of view of the 10 detector to excite the target within the field of view of the detector. The resultant emission beam within the field of view of the detector is received by the beam splitter on its way to the detector. By causing the excitation beam to travel coaxially with the field of view of the detector it improves the overlap volume of the excitation and emission beam detectable by the detector and provides for a more compact device 15 than a multi-static arrangement.

In embodiments, the assembly allows use of the fluorescent properties of certain compounds present in the aquatic environment to measure the concentration of that compound. Examples would be chlorophyll, rhodamine, light oils, fluorescein and 20 others.

Embodiments effectively use a combined excite/detect cube. Previous approaches use separate constructions for excitation and detection whereas embodiments allow for much smaller physical size, and hence provide the possibility of an implementation 25 using multiple instruments to measure many different parameters in one device.

Embodiments use coloured filtering blocks.

In embodiments, the assembly consists of two right angle glass (or other material) 30 prisms, sandwiching a dichroic reflective surface. The source LED is fixed to the first prism part, with the light passing through a suitable filter to eliminate unwanted wavelengths. The sandwiched dichroic mirror reflects the source beam at 90°, with something like 80% efficiency. The reflected beam passes out into the water, where it excites the compound of interest, which then emits light of a different wavelength 35 (fluorescence). The 20% of the light which is not reflected (due to inefficiency of the mirror) is then eliminated by four separate strategies. The second prism is made of coloured glass of the same colour as the emitted fluorescent light. The excitation light that makes it past the mirror is attenuated significantly by this coloured glass, before it

15

encounters a black coating on the far side of the prism, which absorbs much of whatever is left. Any source light that is reflected back from this black surface must then pass back through the coloured prism where further absorption occurs, before reaching the dichroic mirror again, at which point 80% is reflected at 90° to the right (the remainder passing into the original prism and being lost). There is then another pass through the coloured prism with more attenuation, before hitting a detection filter. By this point, most or a large proportion of source excitation light will have been eliminated before reaching the detector which is fixed to that last filter. The fluorescent light meanwhile passes back through the first prism, through the dichroic mirror which does not act on that particular wavelength of light, and through the coloured prism which is designed specifically to allow that colour of light through. Ditto through the final filter, before hitting the detector.

By selection of appropriate filters, glass colours and dichroic mirrors, this could be tailored to allow any combination of excitation and fluorescent wavelengths. It is this versatility combined with the small size of the detection module (being in the region of about 5x5x5mm) is advantageous.

Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

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Claims

1. A monostotic fluorometer, comprising:

a light source operable to transmit an excitation beam;

5 a detector operable to detect emissions; and a beam splitter operable to direct said excitation beam over an excitation path into a fluorescent target to generate said emissions in response to said excitation beam, said beam splitter being operable to receive said emissions over an emission path defined by a numerical aperture of said detector and said beam splitter which is 10 parallel to said excitation path, said emissions passing through said beam splitter and onto said detector.

2. The monostotic fluorometer of claim 1, wherein said light source transmits said excitation beam to said beam splitter over a transmission path and said emissions pass

15 through said beam splitter to said detector over a reception path, said transmission path and said reception path being non-parallel.

3. The monostotic fluorometer of claim 2, wherein said transmission path and said reception path are generally orthogonal.

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4. The monostotic fluorometer of claim 2 or 3, wherein said beam splitter is transparent to said emissions and said reception path is parallel to said excitation path and said emission path.

25 5. The monostotic fluorometer of any preceding claim, wherein said beam splitter comprises a partially reflective mirror.

6. The monostotic fluorometer of any preceding claim, wherein said beam splitter comprises a dichroic mirror.

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7. The monostotic fluorometer of claim 6, wherein said dichroic mirror is transparent to said emissions and said detector is aligned to detect said emissions over said emission path.

35 8. The monostotic fluorometer of any preceding claim, comprising an absorber positioned so that said beam splitter is located between said absorber and said light source.

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9. The monostotic fluorometer of claim 8, wherein said absorber is operable to reduce reflections of any of said excitation beam not directed along said excitation path by said beam splitter which is received by said absorber.

5 10. The monostotic fluorometer of any preceding claim, wherein spatial arrangement of said detector and said beam splitter defines a void which is at least partially filled by a material which provides greater attenuation at frequencies other than a frequency of said emissions.

10 11. The monostotic fluorometer of any preceding claim, comprising a light source filter operable to narrow a bandwidth of said excitation beam.

12. The monostotic fluorometer of claim 11, wherein said light source filter is positioned between said light source and said beam splitter.

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13. The monostotic fluorometer of any preceding claim, comprising a detection filter operable to provide greater attenuation at frequencies other than a frequency of said emissions.

20 14. The monostotic fluorometer of claim 13, wherein said detection filter is positioned between said beam splitter and said detector.

15. The monostotic fluorometer of any preceding claim, comprising a pair of adjacent prisms positioned to orientate said beam splitter.

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16. The monostotic fluorometer of claim 15, wherein hypotenuse surfaces of said pair of prisms are positioned adjacent to each other to orientate said beam splitter.

17. The monostotic fluorometer of claim 15 or 16, wherein said detector is positioned 30 on a cathetus surface of one of said pair of prisms with said light source is positioned on a cathetus surface of another of said pair of prisms.

18. The monostotic fluorometer of any one of claims 15 to 17, wherein said one of said pair of prisms provides greater attenuation at frequencies other than a frequency

35 of said emissions.

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19. The monostotic fluorometer of any preceding claim, comprising a plurality of light sources, each being operable to transmit an excitation beam with a different frequency.

5 20. The monostotic fluorometer of any preceding claim, comprising a plurality of detectors, each being operable to detect emissions with a different frequency.

21. The monostotic fluorometer of any preceding claim, wherein said beam splitter is operable to direct said plurality of excitation beams into said fluorescent target to

10 generate said plurality of emissions and to receive said plurality of emissions, said plurality of emissions passing through the beam splitter onto said plurality of detectors.

22. The monostotic fluorometer of any preceding claim, wherein said light source, said detector and said beam splitter together comprise a detection module, said

15 monostotic fluorometer comprising a plurality of said modules.

23. The monostotic fluorometer of any preceding claim, comprising a fluorescent layer operable to interact with said target, said fluorescent layer having a fluorescence which is modified by properties of said target.

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24. The monostotic fluorometer of claim 23, wherein said properties comprise chemical or physical properties of said target.

25. The monostatic fluorometer of claim 23 or 24, wherein said properties include at 25 least one of a temperature of said target, a pH of the target, an amount of dissolved oxygen in said target and an amount of dissolved carbon dioxide in said target.

26. The monostatic fluorometer of any one of claims 23 to 25, wherein said fluorescent layer is positioned in said excitation path and said layer generates said 30 emissions.

. 27. The monostatic fluorometer of any one of claims 23 to 26, wherein said fluorescent layer is positioned adjacent to one of said pair of prisms.

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35 28. The monostatic fluorometer of any preceding claim, wherein a frequency of said excitation beam is greater than a frequency of said emissions.

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t 29. A method, comprising the steps of:

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transmitting an excitation beam;

detecting emissions; and directing said excitation beam over an excitation path into a fluorescent target to generate said emissions in response to said excitation beam, receiving said emissions over an emission path defined by a numerical aperture of said detector and said beam splitter assembly which is parallel to said excitation path to be detected.

30. A monostatic fluorometer as herein described with reference to the accompanying drawings.

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