GB2523293A

GB2523293A - Method and Apparatus for measuring fluorescence in organisms

Info

Publication number: GB2523293A
Application number: GB1318795.0A
Authority: GB
Inventors: Alan Denton Mortimer; Raphael Skotnicki
Original assignee: VWS UK Ltd
Current assignee: VWS UK Ltd
Priority date: 2013-10-24
Filing date: 2013-10-24
Publication date: 2015-08-26
Also published as: WO2015059452A1; GB201318795D0

Abstract

A method of measuring fluorescence of multi-cellular organisms capable of fluorescing in a first reservoir having an outlet 4 at or near the base of the first reservoir, comprising at least the steps of: (a) providing the multi-cellular organisms 6 in the first reservoir 2 in a liquid 8, said liquid creating a liquid surface 10 across the inside of the first reservoir; (b) providing two or more liquid streams 52 to the liquid surface 10 at or next to the meeting of the first reservoir and the liquid surface, each stream able to create a downward laminar liquid flow 54 along the surface of the first reservoir below the liquid surface and towards the outlet 4; and (c) allowing liquid containing the multi-cellular organisms in the first reservoir to pass out from the first reservoir through the outlet and through a connected fluorescence measuring device. In this way, the multi-cellular organisms are induced by the downward laminar liquid flows to follow their paths towards the outlet, rather than requiring a force to exit the first reservoir.

Description

Method and Apparatus for measuring fluorescence in organisms The present invention relates to a method and apparatus for measuring fluorescence emissions from multi-cellular organisms capable of fluorescing to detect the presence and/or concentration of one or more substances digested by the organism in a liquid.

It is desired to detect specific contaminants and impurities in various types of liquids, usually water, including potable water, waste water, effluent water etc., and at various stages in a process' to determine the efficacy of those processes. The range of impurities detected is not limited and include endocrine disrupters including estrogenic androgenic, thyroid, and/or corticosteroid chemicals.

US2006/0101528A1 mentions that there are a large variety of chemical compounds which diffuse widely in the natural environment, among which are hormonal pollutants in natural water. The presence of these pollutants can be revealed through the observation of fertility problems in various aquatic species. Substances that disrupt the endocrine system mimic the biological effects of hormone factors (oestrogens, androgens and thyroid hormones) which finely regulate many functions such as the functions of homeostasis, etc. The wide spread presence of these factors is causing the attention of authorities about their environmental impact.

A known process uses specific multi-cellular organisms including frog and fish larvae, which can carry fluorescent biomarkers to reveal physiological exposure to contaminants. These particular larvae have been shown to give a very good correlation to predict the effects olthese contaminants in humans.

US200B/0129998A discusses a technique for testing for pollutants in the environment and for pharmaceutical testing. Xenopus tadpoles "light up" (exhibit fluorescence) in response to digesting a pollutant (or drug), and can indicate the presence of several chemical species at the same time. The basic principle involves creating genetically modified constructions that enable a GFP (Green Fluorescent Protein] to be expressed in response to the physiological action of whatever type of molecules a user may be interested in. This molecular dosimeter' is then incorporated in a Xenopus larva, thereby taking into account all the biochemical regulations that can respond in vivo to the sample being tested. For example, if an endocrine disruptor is present, it will activate the response element of various hormones, such as oestrogen or thyroid hormone, triggering the synthesis of fluorescent proteins.

The fluorescence is visible through the transparency of the organism, and can therefore be detected and quantified without sacrificing the animal. The larvae simply need to be placed in the liquid sample to implement the test. The genetic constructions can be altered as required to produce a tailor made range of tests to respond to various disruptive or pharmacological effects. This method combines the advantages of in vivo with the flexibility of in vitro. It rapidly and simply furnishes accurate information of high sensitivity and specificity, together with low cost, economic use of material, and the potential of automation.

US6765656 describes a fountain flow cytometer for this purpose, wherein a sample of fluorescent organisms flows through a flow cell towards a digital camera based on forcing the sample across the camera focal plane. Figures 1 and 2 of the accompanying drawings show this in more detail and are described hereinafter US2008/0129998A requires a pumping subsystem to displace the organisms. The pumping mechanism is intended to cause minimal physical damage and/or stress to the organisms by being based on a pressure and/or gravity differential between a reservoir and a second cyfinder. Figures 3 and 4 of the accompanying drawings show the two particular embodiments described in 11S2008/0129998A. Either an air pipe is used to force a liquid sample with tadpoles from the reservoir and through a tube to the sensor; or a gravity feed arrangement is used to pump the tadpoles between first and second cylinders based on differentiating water levels in the cylinders whose heights are changed relative to a flow cell to achieve pumping of the tadpoles from one container to another.

In addition to the forcing shown in these embodiments, tadpoles and other multi-cellular organisms are also stressed at leaving any relatively large volume and being forced to pass through a narrower conduit or tube, which can make taking measurements of their fluorescence difficult at an optimal liquid flow rate through a flow cell or camera focal plane etc. Thus, currently many images are taken of the tadpoles, few of which are of sufficient quality to be useable.

It is an object of the present invention to provide reduced or minimal stress to multi-cellular organisms passing out of a reservoir and through a flow cell etc., thereby to improve the quality and quantity of fluorescent measurements, and the repeatability of such measurements.

It is also an object of the present invention to provide a system of continuous monitoring of biological parameters revealed by measuring the fluorescent biomarkers emitted within the translucent organisms. This combination of flow-through system with small aquatic fluorescent biosensors, provides a dynamic, near real time, read-out of the quality of liquid samples.

Thus, according to one aspect of the present invention, there is provided a method of measuring fluorescence of multi-cellular organisms capable of fluorescing in a first reservoir having an outlet at or near the base of the first reservoir) comprising at least the steps of: (a) providing the multi-cellular organisms in the first reservoir in a liquid, said liquid creating a]iquid surface across the inside of the first reservoir; (b) providing two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface, each stream able to create a downward laminar liquid flow along the surface of the first reservoir below the liquid surface and towards the outlet; and (cJ allowing liquid containing the multi-cellular organisms in the first reservoir to pass out from the first reservoir through the outlet and through a connected fluorescence measuring device.

In this way, the multi-cellular organisms are induced by the downward laminar liquid flows to follow their paths towards the outlet, rather than requiring a force to exit the first reservoir.

The multi-cellular organisms suitable for use with the present invention include those known in the art, such as those described in 052002/0003625 and US200B/0129998, more particularly frog larvae. Suitable organisms are also described in 052006/0101528 and include at least the Xenopus tadpoles.

The main requirement is that the fluorescent protein is used, and that the strain or other genetic derivation of the organism is characterised by a stable, spatial pattern of fluorescence relating to one or more substances of interest in a liquid, including impurities, pollutants and toxic substances.

Fluorescent proteins are known, and can be chosen from the group comprising fluorescent proteins providing the colours: green, enhanced green, red, blue and indeed yellow. There are also variants of these proteins that change colour with time, or which fuse with other proteins or which are made up of these two fluorescent proteins. The intention of all such proteins is to allow a visual and quantitive analysis. The green fluorescent protein is commonly used, but the fluorescent timc protein is also useful as the change from one colour to another colour reflects the activity of the promoter which directs the expression of the reporter protein, such that it is possible to evaluate the persistence of a compound or of a pollutant on the activity of the promoter.

The multi-cellular organisms are preferably from an amphibian, preferably the frog, and can be selected from Xenopus levies and Xenopus tropicalis. Alternatively, the multi-cellular organisms could be a teleost cell, preferably a zebra fish cell or a medaka cell.

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More generally, the multi-cellular organism can be an aquatic/terrestrial animal such as an amphibian or batrachian, and selected from the Anura, the Urodela and the Apoda, preferably clawed frogs of the families pipidae and ranidae.

The liquid in which the multi-cellular organisms are provided to the fluorescence measuring device is generally water or water based, and is optionally, generally preferably, distinct to the test liquid containing the substance or substances to be analysed and/or quantified by their take-up or digestion by the multi-cellular organisms which causes fluorescence.

The test or sample liquid to be analysed is general also water or water based, although not limited thereto, and can be admixed with the multi-cellular organisms either prior to or as part of step (a) to allow digestion or ingestion of the relevant substance(s) in the test liquid by the multi-cellular organisms, In one arrangement, the test liquid can be provided in the first reservoir and the multi-cellular organisms added thereto. The multi-cellular organisms can then be left for a suitable period of ingestion, such as 1-2 hours, but which may depend on expected pollutant(s) presence or concentration, prior to consideration for passing towards a fluorescence measuring device.

Alternatively, the multi-cellular organisms can be added to the test liquid in a separate container and/or location for a suitable period of lime, prior to their transfer to the first reservoir.

The present invention is not limited by the location or methodology of ingestion of substances from a test liquid by the multi-cellular organisms other than the expectation of the multi-cellular organisms to be capable of fluorescing following ingestion of any relevant substances in the test liquid.

Optionally, the fluorescence measuring device may include one or more Light filters, S such as colour filters, in particular dichroic filters, adapted to selectively pass light of one or more wavelengths whilst reflecting other wavelengths. The or each filter corresponds to the expected wavelength or wavelengths that may fluoresce from the multi-cellular organisms in a manner known in the art Optionally, the method further comprises the step of passing the liquid and multi-cellular organisms through the fluorescence measuring device and into a second reservoir, the second reservoir being operable in the same way as the first reservoir.

That is, the passage of liquid and multi-cellular organisms into the second reservoir creates a liquid surface across the inside of the second reservoir, and there is provided two or more liquid streams to the liquid surface at or next to the meeting of the second reservoir and the liquid surface, able to create a downward laminar liquid flow along the surface of the second reservoir below the liquid surface and towards the outlet of the second reservoir being at or near its base, and allowing liquid containing the multi-cellular organisms in the second reservoir to pass out from the second reservoir and through the outlet.

Optionally, the method of the present invention further comprises the step of passing the liquid and multi-cellular organisms back through the fluorescence measuring device from the second reservoir into the first reservoir.

In this way, the reverse' steps or actions allow a second set of measurements to be taken of the fluorescence of the same multi-cellular organisms shortly after the first set of measurements taken.

Preferably, the method further comprises the step of repeatedly passing the liquid and multi-cellular organisms through the fluorescence measuring device from ftc first reservoir and into a second reservoir and vice versa.

In this way, the method of the present invention provides a simple and efficient method of obtaining a plurality of fluorescence measurements of the multi-cellular organisms, to provide best sampling, both in terms of the quality and quantity of measurements.

As mentioned hereinbefore, either prior to or as part of the method of the present invention, the multi-cellular organisms are provided with a contaminated or otherwise impure test liquid believed to comprise one or more substances or impurities, to ingest or digest said impurities, and whose presence or concentration of a singular or multiple contaminant or pollutant or impurity or drug, etc. is desired to be measured.

Optionally, the method further comprises rinsing the multi-cellular organisms in the first reservoir after the impurity digestion, and prior to passing the multi-cellular organisms through the fluorescence measuring device.

Optionally, the method further comprises repeatedly rinsing the multi-cellular organisms.

Thus, after contaminant or pollutant or impurity or drug digestion) etc., the multi-cellular organisms can be rinsed by the passage of a separate liquid, for example fresh' water, in particular purified water, which liquid is then the basis of the liquid used in the method of the present invention.

The rinsing may be carried out by passing such fresh water through the first reservoir and draining or otherwise disposing through one or more outlets the existing liquid away from the first reservoir, whilst maintaining the multi-cellular organisms in the first reservoir.

Optionally, the method of the present invention further comprises reducing the level of liquid in the first reservoir prior to passing the multi-cellular organism through the fluorescence measuring device.

The reduction at the level of liquid in the first reservoir may be carried out by any suitable process and equipment, generally one or more valves and pumps connected to a suitable outlet. The reduction in the level of liquid in the first reservoir helps to concentrate' the multi-cellular organisms into a smaller volume, preferably with causing any stress thereto, thereby having them closer to the outlet to the connected fluorescence measuring device, as well as having them more frequently at or near to the wall of the first reservoir.

Preferably, the reduction in the level of liquid in the first reservoir is carried out prior to step (b).

Optionally, the height of the liquid in the first reservoir is related to the height of the liquid in a second reservoir when used. As discussed in more detail hereinbelow, optionally the first and second reservoirs are fluidly connected either via the fluorescence measuring device, or by one or more other reservoir outlets, or both, such that there is an intended relationship between the liquid levels in the first and second reservoirs at various stages of the methodology or process used in the present invention.

Preferably, the method further comprises the steps of passing a reference sample of the liquid though the fluorescence measuring device prior to step [a).

In this way, the fluorescence measurements taken can be benchmarked or otherwise referenced or correlated against fluorescent measurements taken of the liquid only.

Optionally, the method of the present invention further comprises one or more of the group comprising: -the step of lowering the flowrate of liquid through the fluorescence measuring device compared with the flowrate of liquid through the outlet of the first reservoir; -the step of dividing the flow of liquid from the outlet of the first reservoir into a first portion to pass through the fluorescence measuring device, and one or more bypass portions not to pass through the fluorescence measuring device; and -the step of taking multiple fluorescence measurements using different imagers of the multi-cellular organisms as they pass through the fluorescence measuring device.

The first reservoir may have any suitable shape, size and dimensions, generally having one or more inlets, optionally a top, and an outlet at or near the base. The reservoir may include one or more inlets and one or more outlets for the passage one or more fluids thereinto and therefrom, whilst at least having one outlet for the passage of liquid containing the multi-cellular organisms out of the reservoir and into a connected fluorescence measuring device.

The two or more liquid streams provided to the liquid surface may be the same or different liquid to the liquid containing the multi-cellular organisms. The liquid of the liquid streams is preferably water or water based, The two or more liquid streams can be provided by a liquid stream provider described in more detail hereinafter. According to another aspect of the present invention, there is provided apparatus to measure the fluorescence of multi-cellular organisms capable of fluorescing comprising at least: (i) a first reservoir having an outlet at or near the base of the first reservoir and containing the multi-cellular organisms in a liquid, said liquid creating a liquid surface across the inside of the first reservoir; (ii) a liquid stream provider able to provide two or more liquid streams to the liquid surlace at or next to the meeting of the first reservoir and the liquid surface, thereby to create downward laminar liquid flows along the surface of the first reservoir below the liquid surface and towards the outlet; (iii) a valve to allow the liquid containing the multi-cellular organisms in the first reservoir to pass through the outlet; and (iv) a fluorescence measuring device connected to the outlet through which the multi-cellular organisms pass.

Preferably, the apparatus further comprises a second reservoir connected to the fluorescence measuring device) the second reservoir also having an outlet at or near its base, and a liquid stream provider able to provide two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface.

Preferably, the or each reservoir has a conical-shaped base with the outlet at the focal point of the cone.

Optionally, the apparatus further comprises a pump to provide direct or indirect movement of the liquid through the fluorescence measuring device. For example, such movement need not be by direct pumping of the liquid as it is passes through the measuring device, but may be instigated by a change of pressure or relative pressure on either side of the measuring device inducing the flow of the liquid therethro ugh.

The or each liquid stream provider may be the same or different, and is not limited by shape, size, design or materials. A number of arrangements can be used to provide the two or more liquid streams used in the present invention. These can include liquid manifotds or distributors in the reservoir having one or more outlets, and able to provide the liquid streams anywhere along the length of the reservoir above the liquid surface. Alternatively, the liquid stream provider comprises one or more pathways located in the reservoir from a suitable liquid source and extending into the reservoir to be at or near the liquid surface.

In one embodiment, the liquid stream provider is a manifold at or near the top of the reservoir, having two or more outlet ports next to the inside surface of the reservoir such that liquid passing through the manifold falls onto the inside surface of the reservoir above the liquid surface, and is able to travel down the reservoir walls towards the liquid surface and thereby to meet the liquid surface at the meeting of the reservoir and the liquid surface.

The liquid streams passing down the inside surface of the reservoir may be the same as or similar to trickles of water passing down a surface such as a window or glass pane. That is, thin or very thin rivulets or streams of liquid acting under gravity to travel downwardly, often not in a straight or direct line.

Optionally, the liquid stream provider is a manifold in a top for the reservoir.

In another embodiment, the liquid stream provider comprises one or more baffles or inner walls within the reservoir down which the liquid streams travel, the ends or bottom of which are at or next to the meeting of the first reservoir and the liquid surface.

in another embodiment, the liquid stream provider comprises a plurality of conduits extending into the reservoir and able to provide a direct pathway fro a liquid stream to the meeting of the first reservoir and the liquid surface.

According to yet another embodiment, the liquid stream provider comprises a plurality of conduits able to provide indirect pathways for two or more liquid streams onto the reservoir and/or onto one or more baffles or inner walls down which the two or more liquid streams can travel towards the meeting of the reservoir and liquid surface.

The two or more liquid streams provided to the liquid surface at or next to the meeting of the reservoir and the liquid surface, create downward laminar liquid flows, which may meet or converge as they travel, along or next to the surface of the reservoir below the liquid surface and towards the outlet. That is, the travel of the liquid streams continues along the surface of the reservoir below the liquid surface in a downward and smooth flow, which is distinct from the remaining body of liquid in the reservoir. The continuance of the liquid streams being provided to the liquid surface continues the downward laminar liquid flow below the liquid surface towards the outlet, and encourages the remaining liquid in the reservoir to pass out of the outlet and into a suitable connection or conduit or other flow path to a connected fluorescent measuring device.

Thus, multi-cellular organisms in the liquid in the reservoir are induced or encouraged to go with the laminar flows of liquid passing through the outlet in a manner of less stress, enabling increased quality and quantity of fluorescence measurements.

The fluorescence measuring device may be any suitable unit or apparatus able to measure fluorescence. Generally this includes a detector, generally a digital detector and possibly including an imager or camera, or including or in combination with one or more filters able to isolate relevant wave lengths of expected fluorescent imaging. Illumination may be provided by suitable light, laser or other light provider or light guide, optionally in combination with one or more mirrors in a manner known in the art.

11S2008/0129998 describes a fountain flow cytometer using a light detector or imager and including a filter and illumination provider by a laser or LED etc. Figure 2 of the accompanying drawings corresponds to Figure 2 of 1JS2008/01 29998, which shows a schematic drawing of an aluminium flow block used as a flow cell having an inlet, an outlet, and a multi-directional pathway there through to achieve a minimal dimension focal plane for its camera.

The fluorescence measuring device of the present invention may include one or more detectors or cameras, being in line or in series or both.

The fluorescence measuring device used in the present invention may also include one or more image detector or camera locations, in particular located below the pathway of the multi-cellular organisms, as it is generally their stomachs or lower abdomen providing the greatest fluorescence. Different imaging angles may be employed to increase the success in image capture and hence more accurate measurement.

The fluorescence measuring device preferably has a singular-directional pathway for the liquid and multi-cellular organism therethrough, so as to minimise stress and direction changes for the multi-cellular organisms, which direction changes are likely to induce stress.

The singular-directional pathway preferably comprises a passageway for the liquid and multi-cellular organisms across the path of one or more image recording devices) and having a deviation of less than 60°. preferably less than 45° or less than 25° between the inlet and the outlet of the fluorescence measuring device.

Optionally, the passageway is wholly or substantially straight', i.e. having no deviation.

The reduction or minimisation of deviation of the passageway between the inlet and outlet of the fluorescence measuring device reduces the stress levels of the multi-cellular organisms, hence increasing the quantity and quality of images ohtainable by the image recording device or devices. This is turn reduces the number of images requires, and the number of passages of the multi-cellular organisms through the fluorescence measuring device to achieve the required measurement of the substances being identified.

The fluorescence measuring device used in the present invention may include one or more changes in the dimensions of the flow path, in particular in a diameter of the conduit or line providing the flow path through the measuring device.

The fluorescence measuring device of the present invention may also involve one or more bypass streams intended to alter the proportion of the liquid flow from the reservoir through the fluorescence measuring device to increase the accuracy, quality and/or quantity of fluorescence measurements by the measuring device, Preferably, the method of measuring fluorescence of multi-cellular organisms in a first reservoir as described herein uses the apparatus as defined herein.

According to a further aspect of the present invention, there is provided a method of passing multi-cellular organisms out of a first reservoir having an outlet at or near the base of the first reservoir, comprising at least the steps of: (a] providing the multi-cellular organisms in the first reservoir in a liquid, said liquid creating a liquid surface across the inside of the first reservoir; (b) providing two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface, each stream able to create a downward laminar liquid flow along the surface of the first reservoir below the liquid surface and towards the outlet; and (c) allowing liquid containing the multi-cellular organisms in the reservoir to pass out from the first reservoir through the outlet.

This aspect of the invention is not limited to fluorescence measuring, but is useable in any arrangement requiring the passage of multi-cellular organisms at least out of a first reservoir in a relatively calm and non-stressed manner.

Optionally, this method further comprises at least one of the further stops of: (I) passing the liquid and multi-cellular organisms into a second reservoir being operable like the first reservoir, (H) passing the liquid and multi-cellular organisms back from the second reservoir into the first reservoir.

[iii) repeatedly passing the liquid and multi-cellular organisms the first reservoir and into a second reservoir, and vice versa.

(iv) rinsing the multi-cellular organisms in the first reservoir prior to passing the multi-cellular organisms out of the first reservoir The present invention is not limited by the number of liquid streams provided to the liquid surface at or next to the meeting of the first reservoir and the liquid surface and where applicable, the number of liquid streams to be provided to the liquid surface at or next to the meeting of the second reservoir and its liquid surface. One embodiment of the present invention is the intention of the liquid streams is to provide a rain effect' that trickles down either along the inside of the reservoir surface or on one or more other surfaces within the reservoir, in order to be provided at or next to the meeting of the reservoir and the liquid surface, so as to provide a liquid flow direction below the liquid surface and along the inside surface of the reservoir that encourages the multi-cellular organisms to transfer in a non-stressed manner towards the outlet.

In one embodiment of the present invention, the method comprises 3, 4, 5 or more liquid streams in step [b), more preferably 4 liquid streams.

The present invention is not limited by the flow rate through the fluorescent measuring device, and/or by the two or more liquid streams provided to the liquid surface of the or each reservoir.

By way of example only, and without limitation, the flow rate of liquid through the fluorescence measuring device could be in the range 1-100ml/rnin, optionally in the range 1050rn1/min, such as 20,30 or 4Oml/min.

Also by way of example only, the total flow rate of the two or more liquid streams in step [bJ could be in the range 50-500m1/min, optionally in the range 80-l5omljmin, such as in the range l00-120m1/min.

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Generally, the flow rate through the fluorescence measuring device will be related to or otherwise complementary to the flow rate of the two or more liquid streams.

The method of the present invention is also able to allow two or more different multi-cellular organisms to be present in the first reservoir, and to pass the different multi-cellular organisms through the fluorescence measuring device simultaneously. That is, either different types of multi-cellular organisms, or the same multi-cellular organisms that are able to digest different substances, ie., impurities, can be provided for measurement at the same time, and using the same methodology, with the apparatus being used to obtain fluorescence measurements from the different types of multi-cellular organisms at the same time. Thus, multi-impurity testing is available to the user, based on a single methodology or apparatus.

Embodiments of the present invention will now be described by way of example only, and with reference to the accompanying schematic drawings in which: Figure 1 is a schematic diagram of an epifluorescent fountain flow cytometer in the

prior art;

Figure 2 is a schematic diagram of an aluminium flow block tube used with the

device of Figure 1 in a prior art;

Figure 3 is a schematic diagram showing a first embodiment of a pumping system in

the prior art of US200B/012993A;

Figure 4 is a schematic diagram illustrating a second embodiment of a pumping

system used in the prior art of US2008/0129998A;

Figure 5 is a schematic diagram of apparatus according to an embodiment of the present invention; Figure 6a and 6b are schematic diagrams of first and second larvae transfer flows in the apparatus of Figure 5; Figure 7 is a part cross section of perspective view of a liquid stream provider for use with the present invention; Figure 8 is a side cross sectional view of first cylinder of Figure 5 with first flow detail; Figure 9 is a perspective part cross sectional view of the first reservoir of Figure 5 showing second flow views; and Figures iDa and lOb are first and second alternatives for changing the flow rate through a measuring device.

Referring to the drawings, Figures 1 -4 are derived from US2008/12998 as illustrative of the prior art. Figure 1 shows a cytometer 100 wherein a sample 102 of fluorescent organisms flows through a flow cell 104 toward the digital camera 106 and optics 108. The cells are illuminated in a focal plane 110 by a laser 112, and an image taken by the CCD camera and lens assembly using a filter 114 that isolates the wavelength of the fluorescence emission.

Figure 2 shows an aluminium flow block used as the flow cell 104 in Figure 1 with input tubing 208 connected to an entrance tube 202 and exit tubing 206. Two vertical holes are drilled in the aluminium flow block as an entrance hole 210 and exit hole 214. As the sample flows up the entrance hole 210 it passes through the focal plane 110 of the camera 106 (not shown). Figure 2 is illustrative of the labyrinthine flow path conventionally used in the art, which causes further stress due because of the meandering direction provided to the organisms through the flow cell 104.

Figure 3 shows a fist embodiment of U32008/0129998. An air pump 412 pumps air into a reservoir 402 to force the tadpoles to he pumped out of the reservoir through pipe 406 and through the flow cell 104 and into a waste container 422.

Whilst the arrangement shown in Figure 3 avoids tadpoles going through ny pump, it can still he seen that forcing the tadpoles through the luhe 406 by the use of air pressure induces stress, which affects the quality and quantity of measurements possible by the flow cell 104.

Figure 4 shows a second embodiment of US2008/0129998A, which uses gravity to pump" the tadpoles from a first cylinder 826 through a flow cell 104 to a second cylinder 806. However, gravity flow is not a natural behaviour for tadpoles, such that their passage through the flow cell is still unnatural to them, and there is still no control over their regularity or evenness through the flow cell.

Figure 5 is a schematic representation of an embodiment of the present invention.

Figure 5 shows apparatus to measure the fluorescence of multi-cellular organisms comprising a first reservoir 2 having an outlet 4 at or near the base of the first reservoir 2, and containing the multi-cellular organisms 6 in a liquid 8, said liquid 8 creating a liquid surface 10 across the inside of the first reservoir 2.

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A liquid stream provider 12, shown and discussed in more detail in relation to Figures 7 and 8, is provided as a top of the first reservoir 2. The first liquid stream provider 12 is able to provide two or more liquid streams, discussed in more detail hereinafter, to the liquid surface 10 at or next to the meeting of the first reservoir 2 and the liquid surface 10, each stream thereby to create a downward laminar liquid flow along the surface of the first reservoir 2 below the liquid surface 10 and towards the outlet 4. A valve 14 is provided to allow the liquid 8 containing the multi-cellular orgamisms 6 in the first reservoir 2 to pass through the outlet 4, and the apparatus includes a fluorescence measuring device 16 connected to the outlet 4 through the multi-cellular organisms 6 and liquid 8 can pass.

The apparatus shown in Figure 5 further comprises a second reservoir 20 connected Lo the fluorescence measuring device 16, the second reservoir 20 also having an outlet 22 at or near its base) and a second liquid stream provider 24, as part of the top of the second reservoir 20. Each of the first and second reservoirs 2, has a conical-shaped base, with their respective outlets 4, 22 being at the focal point of the cone. The apparatus further comprises a pump 26 to provide movement of the liquid through the fluorescence measuring device 16.

In use, a fluid under test can pass into the first container 2 via a strainer, a number of regulators, valves and heaters and flow gauges etc. generally shown in Figure 5 as item 30, into the first reservoir 2 containing the multi-cellular organisms 6 such as fog larvae. Such frog larvae have a GTP which can be excited to emit fluorescence upon the digestion of a particular substance, for example being an impurity or contaminant or pollutant in the test fluid, in a manner known in the art Optionally, the test fluid is maintained in the first reservoir 2 for a defined period of time. Alternatively, the test fluid passes through the first reservoir 2 for a period of time and out to a drain 34 or otherwise. The period of time may vary depending upon the nature of the multi-cellular organisms and the intended contaminant, etc. to be detected, and the skilled man will be aware of these requirements.

Prior to the introduction of the multi-cellular organisms and contaminated test fluid, a reference sample of the liquid to be used for the passage of the multi-cellular organism 6 through the fluorescence measuring device 16 can pass through the first reservoir 2 and through the fluorescence measuring device 16 to provide a referencing or benchmarked series of measurements. This can reduce and unexpected of artificial measurements caused by fluorescence readings of the liquid

IC

iiseit.

Once the user considers that there has been sufficient digestion of the or each relevant substance by the multi-cellular organisms, the contaminated fluid can be drained away and a rinsing fluid such as water, preferably purified water, can be provided through the same entry mechanism 30 and into the first reservoir 2.

Alternatively, the rinsing fluid is provided through the second reservoir 20 and back through the fluorescence measuring device 16 into the first reservoir 2, whilst the first reservoir is drained through a pumped outlet 32 using the pump 26. The flow rate for rinsing fluid could he relatively high, such as up to BOo mI/mm.

Figure 5 shows the pump 26 having both forward and reverse flow operations, and connected via four uni-directional valves 14 able to provide different flow arrangements between the first and second reservoirs 2, 20, and indeed one or more drains 34, so as to provide any desired flow arrangements thereinbetween.

To reduce any possible effect from the contaminated test fluid, the multi-cellular organisms 6 can be rinsed more than once using the same or different rinsing fluid and/or rinsing operations, such that the multi-cellular organisms are then present in a final liquid 8 for use in the method of measuring fluorescence according to the present invention.

Figure 6a shows a first larvae transfer flow using the apparatus of Figure 5.

Optionally, the leveL of liquid 8 in the first reservoir 2 is reduced through the pumped outlet 32, and through one or more of the valves 14, to a drain 34.

The reduction of liquid 8 as shown in Figure 6a compared to Figure Shelps concentrate the multi-cellular organism 6 into a smaller volume, such that the subsequent step has greater effect on the multi-cellular organisms 6 due to their increased concentration at or next to the sides of the first reservoir 2.

in Figure 6a, the pump 26 is operated to take liquid Ba in the second reservoir 20 through two of the valves 14 and into the first reservoir 2. Naturally, the reduction of the amount and therefore height of liquid 8a in the second reservoir 20 starts to draw the liquid B in the first reservoir 2 through the fluidly connected fluorescence measuring device 16.

in the embodiment of the method of the present invention shown iii Figure 6a, an inlet stream 36 from the pump 26 is provided into the first liquid stream provider 12 via a top inlet port 38, and then as two or more liquid streams to the liquid surface 10 at or next to the meeting of the first reservoir 2 and tile liquid surface 10.

Referring to Figure 7, 8 and 9, Figure 7 shows a part cross-sectional view of the first liquid stream provider 12, having an inlet port 38 in the top thereof, and internafly four symmetrically arranged pipings 40 towards four outlet ports 42 around a lower vertical surface 44 of the top 12.

Figure 8 shows a side cross sectional view of the first reservoir 2, and flow paths for fluid through the top 12 and the first reservoir 2. Figure 8 shows a flow path 48 for the inlet of fluid, such as the fluid 8a from the second reservoir 20 provided via the pump 26, into the first liquid stream provider 12 and through its pipings 40. From their outlet ports 42, a flow path of the fluid 50 falls onto the inner wall of the first reservoir 2 and flows theredown in a manner of a small trickle or small stream, depending upon the flow rate, in a manner of rain down a surface, until such flow 52 meets the liquid surface 10 at or next to the meeting of the first reservoir 2 and the liquid surface 10. The momentum of the downward flow 52 then creates downward laminar liquid flows 54 along or next to the downward surface of the first reservoir 2 below the liquid surface 10, and towards the outlet 4.

Figure 9 provides more illustration of the flow paths 52 after they fall onto the reservoir inner surface, being a non-regular passage as they travel down the inside of the vessel under 2 prior to hitting the liquid surface 10, from which their flow is more laminar as they continue to travel down the inside of the first reservoir 2. The first liquid stream provider 12 is not shown in Figure 9.

The effect of the flow paths 52 and subsequent downward flows for 54 encourages the remaining liquid 8 in the first reservoir 2 to flow down towards the outlet 4 in a less turbulent manner, and thereby encourages the larvae 6 to travel with the flow created iii the first cylinder 2 towards the outlet 4. Without significant turbulence, the larvac 6 are non-stressed, and can thereby he induced to pass out of the outlet 4 in the most calm manner desired prior to their passage towards and through the fluorescence measuring device 16.

The larvae 6 travel through the fluorescence measuring device 16 and into the second reservoir 20, as started to be shown in Figure 6a.

Once a sufficient number are collected in the second reservoir 20, as shown in Figure 6b, the reverse operation can be carried out. That is, the pump 26 is reversed, and different uni-directional valves 14 are used to transfer liquid 8 from the second reservoir 2 through the pump 26 and into the second reservoir 20 to draw liquid 8a in the second reservoir 20 through the fluorescence measuring device 16.

The second reservoir 20 has a second Uquid stream provider 24, able to provide the same liquid stream arrangement as discussed in relation to Figures 7-9 to the second reservoir 20, so as to provide two or more, generally four, liquid streams to the liquid surface at or next to the meeting of the second reservoir 20 and the liquid surface therein. This creates downward laminar liquid flows down along or next to the surface of the second reservoir 20 below the liquid surface and towards its outlet 22. From the outlet 22, the liquid Ba and larvae 6 pass hack through the fluorescence measuring device 16 and into the first reservoir 2, in a manner shown in Figure 6b.

Thus, in the same way as described above, operation of the second liquid stream provider 24 provides laminar liquid flow to the outlet 22, to encourage non-turbulent flow of the remainder of liquid Ba in the second reservoir 20 towards the outlet 22, thereby inducing and encouraging the larvae 6 in the second reservoir 20 to pass through the outlet 22 in the most calm and non-stressful manner, prior to their passage through the fluorescence measuring device 16 in the same way as described hereinabove.

The second larvae transfer as shown in Figure 6b results in the location of at least the majority of the larvae 6 in the first cylinder 2 as shown in Figure 6a, Thereafter, a repeated operation of the steps as described above, allows the user to pass the larvae 6 through the fluorescence measuring device 16 a number of times between the first and second reservoirs 2, 20, in order to take a plurality of fluorescence measurements in an easy and rapid and repeatable manner.

The fluorescence measurements taken by the fluorescence measuring device 16 can then be interpreted using analysis software known in the art, to be correlated with the levels of the substance or substances in the impure test liquid first passed to the larvae 6. The method of the present invention can be automated, and the same or different impurity streams provided at regular intervals to the larvae in order to determine changes and/or differences between contaminants, impurities, etc. in different impurity streams.

Figure ba shows a first arrangement having a single-directional or straight conduit for the passage of a larvae 6 through the fluorescence measurement device 16, the larvae 6 and liquid Ba travelling generally from right to left', for example from the second reservoir 20 in the embodiments of the present invention described above, to the first reservoir 2.

As a sensor (not shown) confirms the approach of the frog larvae 6 towards the focal plane 62 of a camera or detector [not shown) below the conduit 60, it is desired to slow or reduce the flow rate of the liquid Ba in the conduit 60 to provide more time for the camera to focus and take a better fluorescence image, reading or measurement of the larvae 6. For this, a counter-current 64 can be provided against the flow or current of the liquid 8a, at a relative flow rate or speed calculated to provide the desired flow rate of the frog larvae 6 particularly above the focal plane 62.

Figure lOb shows an alternative arrangement to that in Figure iOa, wherein the liquid flow 8a in the conduit 60a, prior to the focal plane 62 of the camera or imager [not shown), is divided into a main stream 66 still carrying the frog larvae 6, and two bypass streams 68 able to take some of the liquid Ba from the entry end of the conduit 6th to the other end of the conduit 60b, thereby reducing the flow rate of the main stream 66, and inherently of the frog larvae 6, at the focal point 62.

In general, the present invention provides a method and apparatus able to reduce or minimise stress to multi-cellular organisms passing out of a reservoir and through a flow cell etc. This clearly improves the quality and quantity of fluorescent S measurements able to be taken, and the repeatability of such measurements, by allowing the measurement device to take a better, clearer and optionally slower fluorescence image, reading or measurement of the multi-cellular organisms This increases the quality and quantity of Pollutant measurement information from the images obtained, In particular, the present invention allows a user to set up a system of continuous monitoring of biological parameters revealed by measuring the fluorescent biomarkers emitted within the translucent organisms. This combination of flow-through system with small aquatic fluorescent biosensors, provides a dynamic, near real time', read-out of the quality of liquid samples being tested:

Claims

Claims 1. A method of measuring fluorescence of multi-cellular organisms capable of fluorescing in a first reservoir having an outlet at or near the base of the first reservoir comprising at least the steps of: (aJ providing the multi-cellular organisms in the first reservoir in a liquid, said liquid creating a liquid surface across the inside of the first reservoir; (b] providing two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface, each stream able to create a downward laminar liquid flow along the surface of the first reservoir below the liquid surface and towards the outlet; (c) allowing liquid containing the multi-cellular organisms in the reservoir to pass out from the first reservoir through the outlet and through a connected fluorescence measuring device.
2. A method as claimed in claim 1 further comprising admixing the multi-cellular organisms with a test liquid to be analysed either prior to or as part of step (a] to allow digestion of any substances in the test liquid by the multi-cellular organisms.
3. A method as claimed in claim 1 or claim 2 further comprising rinsing the multi-cellular organisms in the first reservoir prior to passing the multi-cellular organisms through the fluorescence measuring device.
4. A method as claimed in claim 3 comprising repeatedly rinsing the multi-cellular organisms prior to passing the multi-cellular organisms through the fluorescence measuring device S. A method as claimed in any one of the preceding claims further comprising reducing the level of liquid in the first reservoir prior to prior to passing the multi-cellular organisms through the fluorescence measuring device.6. A method as claimed in any one of the preceding claims further comprising the step of passing the liquid and multi-cellular organisms through the fluorescence measuring device and into a second reservoir, the second reservoir being operable in the same way as the first reservoir.7. A method as claimed in claim 6 further comprising the step of passing the liquid and multi-cellular organisms back through the fluorescence measuring device from the second reservoir into the first reservoir.8. A method as claimed in claim 7 further comprising the step of repeatedly passing the liquid and multi-cellular organisms through the fluorescence measuring device from the first reservoir and into the second reservoir and vice versa.9. A method as claimed in any one of claims 6 to 8 wherein the height of the liquid in the first reservoir is related to the height of the liquid in the second reservoir.10. A method as claimed in any one of the preceding claims further comprising the step of passing a reference sample of the liquid though the fluorescence measuring device prior to step (a].11. A method as claimed in any one of the preceding claims further comprising the step of lowering the flowrate of liquid through the fluorescence measuring device compared with the flowrate of liquid through the out:Iet of the first reservoir.12. A method as claimed in any one of the preceding claims further comprising the step of dividing the flow of liquid from the outlet of the first reservoir into a first portion to pass through the fluorescence measuring device, and a bypass portion not to pass through the fluorescence measuring deWce.13. A method as claimed in any one of the preceding claims further comprising the step of taking multiple fluorescence measurements of the multi-cellular organisms as they pass through the fluorescence measuring device.14 A method as claimed in any one of the preceding claims comprising 3, 4, 5 or more liquid streams in step (bJ.15. A method as claimed in any one of the preceding claims further comprising the step of providing two or more different multi-cellular organisms in the first reservoir and passing all the multi-cellular organisms through the fluorescence measuring device simultaneously.16. Apparatus to measure the fluorescence of multi-cellular organisms capable of fluorescing comprising at least: fi) a first reservoir having an outlet at or near the base of the first reservoir and containing the multi-cellular organisms in a liquid, said liquid creating a liquid surface across the inside of the first reservoir; (ii) a liquid stream provider able to provide two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface thereby to create downward laminar liquid flows along the surface of the first reservoir below the liquid surface and towards the outlet; [iii) a valve to allow the liquid containing the multi-cellular organisms in the first reservoir to pass through the outlet; and (iv) a fluorescence measuring device connected to the outlet through which the multicellular organisms pass.17. Apparatus as claimed in claim 16 further comprising a second reservoir connected to the fluorescence measuring device, the second reservoir also having an outlet at or near its base, and a liquid stream provider able to provide two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface.18 Apparatus as claimed in claim 16 or claim 17 wherein the or each reservoir has a conical-shaped base with the outlet at the focal point of the cone.19. Apparatus as claimed in any one of claims 16 to 18 further comprising a pump to provide movement of the liquid through the fluorescence measuring device.20. Apparatus as claimed in any one of claims 16 to 19 wherein the or each liquid stream provider is a manifold having two or more outlets ports, each port being able to provide a liquid stream.21. Apparatus as claimed in claim 20 wherein the or each liquid stream provider is a manifold in a top for the reservoir.22. Apparatus as claimed in any one of claims 16 to 21 wherein the fluorescence measuring device comprises an image recording device located below the passage of the multi-cellular organisms through the fluorescence measuring device.23. Apparatus as claimed in any one of claims 16 to 22 wherein the fluorescence measuring device comprises one or more selected from the group comprising: -a change in the dimensions of the passage of the liquid and multi-cellular organisms through the fluorescence measuring device; -a change in the flow rate of the liquid and multi-cellular organismS s through the fluorascence measuring device; -a passageway across an image recording device having a deviation of <60, preferably c45Q or c25, between the inlet and the outlet of the fluorescence measuring device; and -a change in the amount of passing through the fluorescence measuring device.24. A method of measuring fluorescence of multi-cellular organisms in a first reservoir using the apparatus as defined in any one of claims 16 to 23.A method of passing multi-cellular organisms out of a first reservoir having an outlet at or near the base of the first reservoir, comprising at least the steps of (a) providing the multi-cellular organisms in the first reservoir in a liquid, said liquid creating a liquid surface across the inside of the first reservoir; (b) providing two or more liquid streams to the liquid surface at or next to the meeting of the first reservoir and the liquid surface, each stream able to create a downward laminar liquid flow along the surface of the first reservoir below the liquid surface and towards the outlet; and (c) allowing liquid containing the multi-cellular organisms in the reservoir to pass out from the first reservoir through the outlet.26. A method as claimed in claim 25 further comprising at least one of the further steps of: (i) passing the liquid and multi-cellular organisms into a second reservoir being operable like the first reservoir, (ii) passing the liquid and multi-cellular organisms back from the second reservoir into the first reservoir.(iii] repeatedly passing the liquid and mu1ticellular organisms through the fluorescence measuring device from the first reservoir and into a second reservoir1 and vice versa.(iv] rinsing the multi-cellular organisms in the first reservoir prior to passing the multi-cellular organisms out of the first reservoir [v] repeatedly rinsing the muIti-celluar organisms.(vi] passing a reference sample of liquid though the fluorescence measuring device prior to step (a).