CA2136209A1 - Apparatus and method for inducing and detecting fluorescence - Google Patents
Apparatus and method for inducing and detecting fluorescenceInfo
- Publication number
- CA2136209A1 CA2136209A1 CA002136209A CA2136209A CA2136209A1 CA 2136209 A1 CA2136209 A1 CA 2136209A1 CA 002136209 A CA002136209 A CA 002136209A CA 2136209 A CA2136209 A CA 2136209A CA 2136209 A1 CA2136209 A1 CA 2136209A1
- Authority
- CA
- Canada
- Prior art keywords
- light
- sensor
- fluorescence
- detector
- fluid medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6473—In-line geometry
- G01N2021/6476—Front end, i.e. backscatter, geometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6484—Optical fibres
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N21/85—Investigating moving fluids or granular solids
- G01N21/8507—Probe photometers, i.e. with optical measuring part dipped into fluid sample
Abstract
An apparatus and a method for inducing and detecting fluorescence in a fluid medium containing at least one fluorophore. The apparatus comprises a light emitter adapted to emit light of a wavelength capable of exciting fluorophores in the fluid medium, a light detector adapted to detect fluorescence emitted by the fluorophores excited by the exciting light, a sensor body, preferably being solid and made of quartz, having internally reflecting wall parts capable of reflecting at least light having a wavelength corresponding to the wavelength of the fluorescence and having a sensor face adapted to be exposed to the fluid medium, the sensor body being adapted to receive exciting light from the light emitter, such as via an optical fiber, and to transmit the received exciting light into the fluid medium through the sensor face, to receive, through the sensor face, fluorescence emitted by excited fluorophores in the fluid medium, and to transmit at least part of the received fluorescence to the light detector, both the light emitter and the detector being positioned in a distance from the sensor face, and the relative positioning of the light emitter, the optical detector, and the sensor face being such that the detector is able to receive light transmitted from at least a portion of that part of the sensor face which receives light from the light emitter. The apparatus is especially well suited for operation in turbid or highly turbid media such as a fermentation tank or a wastewater in a wastewater purification plant.
Description
2 ~1~ 3 ~
APPARATUS AND METHOD FOR INDUCING AND DETECTING FLUORESCENCE
The present invention concerns a novel apparatus and method --for inducing and detecting fluorescence. The apparatus is particularly useful for measuring fluorescence emitted from 5 turbid or highly turbid fluid media, such as, e.g., fluo- -rescence emitted from living cells in a fermentation tank or -in a mixed culture in a water purification plant. Thus, the apparatus is most valuable f~or performing the fluorescence determinations in the method disclosed in W090/10083 published 7 September, 1990, which relates to a method for controlling and/or optimising a process in which an aqueous system~comprising biodegradable material, such as waster j ~
water~or oewage, is subjected to biologica1 treatment using , -mixed cultures of microorganiæms.
~15 ~ US 4~.~577~.~110 discloses an~apparatus for inducing 1uorescence j ':7 ~in,~and~ asuring~fluorescence emitted rom, a biological !
medium.`~The~apparàtus is adapted to~launch, from a continuous light source,~a~beam~of~exciting light through a ring-shaped detector.~ The~aplparatus~is designed so that the field of 20 ~illu ination~of the exciting light is substantially contained ; within~the~field of view of the detector measuring the ~ ~ emitted fluorescence.
; ~ ~EP 0.04~7.094~discloses an apparatus for inducing and detec-tIng~f1uorescence~in~a medium.~The pulsed exciting light and j 25 ~the~fluoresc-nce~`;emitted~from~the medium are guided to and ! -from~the~easured~medium by~means of optical fibres. ! `
In highly turbid media, light is not able to travel more than a few mm~or even only~fractions of a mm before it becomes very considerably attenuated. Therefore, fluor-scence 30 mea~surements~in~such media present severe problems, and there F`
is a~considerable~demand for fluorescence measuring equipment which~is able to perform measurements with a high sensitivity ;
and over~a broad dynamic range.
- ~
'`: ~
2 ~ 3 ~
The present invention relates to an apparatus of a novel type which is particularly adapted for performing the inherently very difficult fluorescence measurements in highly turbid media, but which will also be advantageous for applications in less turbid or non-turbid media.
DISCLOSURE OF THE INVENTION
In one aspect, the invention relates to an apparatus for inducing and detecting fluorescence in a fluid medium containing at least one fluorophore, the apparatus ~lO comprising:
- a light emitter adapted to emit light of a wavelength capable of exciting fluorophores in the fluid medium, - a light detector adapted to detect fluorescence emitted by the fluorophores excited by the excitlng light, lS - ~ a sensor body having internally reflecting wall parts capable of reflecting at least light having a wavelength corresponding to the wavelength of the fluorescence and having a sensor face adapted to be exposed to the fluid medium, the sensor body being adapted to receive exciting light from the light emitter, and to transmit the received exciting light into the fiuid medium through the sensor face, to - receive, through the sensor face, fluorescence emitted by excited fluorosphores in the fluid medium, and to transmit at least part of the received fluorescence to the light detector, both the light emitter and the detector being positioned in a distance-from the sensor face, and the relative positioning of the light emitter, the optical detector, and the sensor `
face being such that the detector is able to re~e-ive~-light transmitted from at least a portion of that part of the i~ :
sensor face which receives light from the light emitter.
, ~ .
~. ~
W093/23738 ~` PCT/D~93/00171 In another aspect, the invention relates to a method for ~-inducing and detecting fluorescence in a fluid medium containing at least one fluorophore, the method compri4ing: ~
- transmitting light from a light emitter through a sensor body having internally reflecting wall parts at least capable of reflecting light having a wavelength corresponding to the wavelength of the fluorescence and through a sensor face thereof into the fluid medium, at least part of the light being of a wavelength capable of exciting the fluorophores so as to induce the emission of fluorescence, - receiving, through the sensor face, fluorescence emitted by the excited fluorophores in the fluid medium, and -transmitting at least part of the received fluorescence through the sensor body to a light detector adapted to detect the fluorescence emitted by the fluorophores excited by the exciting light, i at least part of the fluorescence transmitted to the detector being fluorescence received through a part of the sensor face 20 from which }ight from the light emitter is emitted to the fluid medium.
The fluid medium will normally be a liquid, but it may also be a mixture of a liquid and a gas, or it may be a gas with, e.g. suspended particles containing fluorophores.
25 The term "fluorophore" is to be understood in its accepted meaning as a substance which, when excited by absorption of electromagnetic irradiation (light), will decay to a lower - energy state with emission of electromagnetic radiation r having a wavelength longer than the wavelength of the ~- 30 exciting electromagnetic irradiation. Many important fluo-- rescent substances may be excited with Ultra Violet light and will decay with emission of light in the visual range, but ~there are also substances which both absorb and emit light in 2 ~ 3 t i; ? ~
W093~23738 PCT/DK93/00171 the Ultra Violet region, as well as substances which both absorb and emit light in the visual region, and substances which emit in the Near Infra Red-region. The apparatus according to the present invention can be used for measurements in connection with any of these types of substances, although it will be described in the following with particular emphasis on measurements on substances which absorb in the Ultra Violet region and emit visual light.
A central feature of the apparatus of the invention is the sensor body, which constitutes a particularly advantageous functional interface between on the one hand the light emitter and the light detector and on the other hand the fluid medium. One function of the sensor body is to establish such a geometrical position relation between on the one hand the liquid medium and on the other hand the light emitter and the detector that the detector is capable of receiving light from the same part of the liquid medium as has been excited by illumination with the exciting light. The importance of this relation resides in the fact that in a highly turbid medium, the available fluid volume which can be excited is represented by a layer having a thickness of only a few mm, or even only a fraction of a mm, immediately in front of the sensor face and that any part of this layer which has not ``
received the exciting light will not contribute to the active measuring volume.
Thus, in a preferred embodiment, the relative positioning of~
the light emitter, the sensor face, and the optical detéctor ~
is such that light emitted by the light emitter is able to directly illuminate the predominant part of the sensor face.
On the other hand, as the wall parts are capable of`
reflecting the exciting light, any part of the emitted light which is not directly directed to the sensor face-~i-ll~-be transmitted thereto through reflection on the walls.
In another preferred embodiment made possible through the use of the sensor body interface, the relative positioning of the 2 ~ 3~?~
light emitter, the sensor face, and the optical detector is such that the detector is able to directly receive light from a predominant proportion of the illuminated part of the sensor face. Reflection on the walls of the sensor will then further enhance the amount of fluorescence which becomes available for detection.
The exciting light is preferably pulsed. This gives several advantages. Thus, pulsing makes it possible to compensate for any background signal in the measurement. In some of the important types of measurements on fluids containing biological material, e.g., in purification plants, day-light may generate an error signal, which can be compensated for by subtracting the background signaL from the peak fluorescence signal following excitation. Another important advantage of the usè of pulsed induction of fluorescence is that also the fluorescence generated is measured as a pulsed signal, whereby the peak intensity may be orders of magnitude higher 1~`
than when exciting the fluid medium with continuous light having the same mean intensity. Thus, when pulsing the output of the light source, the peak intensity of the light pulses may correspond to the intensity emitted from a continuous 50-~- ~ lOO kW light source.
, When using a pulsed light source, care -should be taken to shield the detecting electronics from noise generated by the light source and its controlling electronics. An advantageous way of reducing the noise due to the pulsed light source is - to separate the pulsing and the detecting electronics physically. This may be accomplished by the use of fibre optics. Thus, in a preferred embodiment of the apparatus according to the invention, the li~ht emitter is a light ~ emitting end of a fibre optical means, such as an optical -~ fibre bundle, having a light receiving end adapted to receive ~- light from a light source.
.
~ The fact that an optical fibre emits light from a very small : ~ ~35 area and at a maximum angle determined by the fibre material -~
2 ~ ~ n ,~
and the wavelength of the light, the maximum angle being smaller for Ultra Violet-transmitting fibres than for fibres transmitting visual light, is compensated for by the fact that the light emitting end of the fibre optical means is positioned at a distance from the sensor face. As will be explained in greater detail in connection with the drawing, this configuration makes it possible for the fibre optical means to illuminate and thus excite a satisfactorily large area of a turbid medium.
It is believed that the special way of using fibre optics - where the light emitting end thereof is kept at a distance from the sensor face, thus enlarging the area of illumination `
and at the same time permitting a detector to "see" all or a considerable proportion of the illuminated area is novel E~E
lS se, and thus, a particular aspect of the present invention can be expressed as an apparatus for inducing and measuring fluorescence in a fluid medium containing at least one fluorophore, the apparatus comprising:
a light emitter adapted to emit light of a wavelength able to excite fluorophores in the fluid medium, - optical fibre means adapted to receive and guide exciting light from the light emitter and having a light emitting end, - a light detector adapted to detect fluorescence emitted by the fluorophores excited by the exciting light, - a sensor body having a sensor face adapted to be exposed to the fluid medium, - the sensor body being adapted to receive exciting-light - ~
; from the light emitting end of the optical fibre means, and~
to transmit the received exciting light into the fluid medium, to receive fluorescence emitted by excited fluorosphores in the fluid medium, and to transmit at~least part of the received fluorescence to the light detector,~
the sensor face being positioned in a distance from-~he-l-ight emitting end of the optical fibre means, and the relative positioning of the light emitting end of the optical fibre means, the sensor face, and the optical detector being such that light emitted by the optical fibre means is able to ~1 3fi .~.~`.?
W093~23738 PCT/DK93/00171 illuminate a part of the sensor face, and such that the detector is able to receive light from at least a part of the thus illuminated part of the sensor face.
Depending on the particular measurement to be performed, the S pulsing may be performed with time intervals varying from fractions of a second to the order of minutes or hours. The duration of each single pulse is normally of the order of fractions of a second.
Alternatively, in a preferred embodiment of the present invention, a measurement may be performed by transmitting a series of pulses, such as 12 to 200 pulses, measuring the generated fluorescence in the peaks and between the peaks and assigning the mean value of the measured fluorescence to be the actual value. The pulses in the measurement may in principle be generated at any frequency; at present, a frequency of in the order of 4 Hz is preferred. Measurements may be performed at regular intervals, such as lO to 60 times or~more an hour. Naturally the frequency of the pulses in the measurement, the number of pulses in the measurement and the 20~ time interval in which the measurements are performed is adapted to each other. If e.g. 180 measurements are performed an hour, the number of pulses used for each measurement is usually in the order of 4-32.
- ~ ;
For the generation of the exciting light, a wide variety of-~ 25 light sources may be used, ranging from Ultra Violet sources to broad-band sources, depending on the properties of the fluorophores to be measured. For most applications, the exciting light will be light in the Ultra Violet region.
~ The wavelength range of the exciting light is normally ~ 3~0 limited, e.g., by incorporating, in the light emitter, light filtering means permitting the light emitter to emit filtered light. In a preferred embodiment, the filtering means is placed between the light source and the light receiving end of the fibre optics.
' ' ,,,,,,...... .. - -- - - -The sensor body is normally a monolithic body, but it is possible, and within the scope of the present invention, to establish the function of the sensor body by combination of `
two or more individual parts which together constitute the S sensor body. The sensor body may be a hollow body, which may present the advantage that the amount of material in the light path which could potentially generate fluorescence (most materials are capable of some degree of fluorescence) is reduced, but for most practical purposes, it is preferred that the sensor body is solid. The use of a solid sensor body has the advantage that a compact and robust design can be obtained, and that the danger of formation of water of '`~
condensation at critical internal parts of the apparatus is obviated or considerably reduced.
The sensor body may be made of any material which is capable of effectively transmitting the exciting light and the fluorescence, such as quartz or sapphire when the exciting light is in the Ultra Violet region,' or glass when the exciting light is in the visual'range. Due to the exciting ~20 light travelling in the sensor body, fluorescence from the material constituting the sensor body may occur, depending on the material of the sensor body. Therefore, it is important to select a~sensor body material which will emit as little fluorescence as possible. For most cases, this means that a high purity of the sensor body material should be secured, as impurities will tend to increase the amount of fluorescence.
It is preferred that the sensor body has a substantially ~~ - '~
symmetrical cross section in a direction transverse to the `' `
' 30 sensor face. In this connection, "transverse" should not be understood as limited to "perpendicular to", but rather --includes both the perpendicular relationship and any slanted or oblique relationship. A suitable method of producing- a`'- -sensor body of the kind disclosed herein is to cut a piece`~of a rod of the material in question, e.g. a quartz rod, and subjecting the piece to working and polishing so as to bring it into a desired shape and establish end faces suitable as ~3 ~ rj I
W093t23738 PCT/DK93/00171 sensor face and face receivlng the detector and the light emitter, respectively.
The cross section of the sensor body may, e.g., be substan-tially elliptical, in particular substantially circular.
S The reflective property of the wall parts of the sensor body are suitably obtained due to a coating of the wall parts. The coating may be any suitable coating resulting in the desired reflecting properties, such as a coating selected from dielectric coatings and metal coatings. The method of application of the coating will be adapted to the coating material and may comprise application from a liquid or from a vapour. In a presently preferred embodiment, the sensor body is coated with an external coating with aluminum applied by vapour deposition.
lS The sensor body will normally be an elongated body.
The dimensions of the sensor body may vary over a wide range.
Normally, the sensor body will have a length in the range of
APPARATUS AND METHOD FOR INDUCING AND DETECTING FLUORESCENCE
The present invention concerns a novel apparatus and method --for inducing and detecting fluorescence. The apparatus is particularly useful for measuring fluorescence emitted from 5 turbid or highly turbid fluid media, such as, e.g., fluo- -rescence emitted from living cells in a fermentation tank or -in a mixed culture in a water purification plant. Thus, the apparatus is most valuable f~or performing the fluorescence determinations in the method disclosed in W090/10083 published 7 September, 1990, which relates to a method for controlling and/or optimising a process in which an aqueous system~comprising biodegradable material, such as waster j ~
water~or oewage, is subjected to biologica1 treatment using , -mixed cultures of microorganiæms.
~15 ~ US 4~.~577~.~110 discloses an~apparatus for inducing 1uorescence j ':7 ~in,~and~ asuring~fluorescence emitted rom, a biological !
medium.`~The~apparàtus is adapted to~launch, from a continuous light source,~a~beam~of~exciting light through a ring-shaped detector.~ The~aplparatus~is designed so that the field of 20 ~illu ination~of the exciting light is substantially contained ; within~the~field of view of the detector measuring the ~ ~ emitted fluorescence.
; ~ ~EP 0.04~7.094~discloses an apparatus for inducing and detec-tIng~f1uorescence~in~a medium.~The pulsed exciting light and j 25 ~the~fluoresc-nce~`;emitted~from~the medium are guided to and ! -from~the~easured~medium by~means of optical fibres. ! `
In highly turbid media, light is not able to travel more than a few mm~or even only~fractions of a mm before it becomes very considerably attenuated. Therefore, fluor-scence 30 mea~surements~in~such media present severe problems, and there F`
is a~considerable~demand for fluorescence measuring equipment which~is able to perform measurements with a high sensitivity ;
and over~a broad dynamic range.
- ~
'`: ~
2 ~ 3 ~
The present invention relates to an apparatus of a novel type which is particularly adapted for performing the inherently very difficult fluorescence measurements in highly turbid media, but which will also be advantageous for applications in less turbid or non-turbid media.
DISCLOSURE OF THE INVENTION
In one aspect, the invention relates to an apparatus for inducing and detecting fluorescence in a fluid medium containing at least one fluorophore, the apparatus ~lO comprising:
- a light emitter adapted to emit light of a wavelength capable of exciting fluorophores in the fluid medium, - a light detector adapted to detect fluorescence emitted by the fluorophores excited by the excitlng light, lS - ~ a sensor body having internally reflecting wall parts capable of reflecting at least light having a wavelength corresponding to the wavelength of the fluorescence and having a sensor face adapted to be exposed to the fluid medium, the sensor body being adapted to receive exciting light from the light emitter, and to transmit the received exciting light into the fiuid medium through the sensor face, to - receive, through the sensor face, fluorescence emitted by excited fluorosphores in the fluid medium, and to transmit at least part of the received fluorescence to the light detector, both the light emitter and the detector being positioned in a distance-from the sensor face, and the relative positioning of the light emitter, the optical detector, and the sensor `
face being such that the detector is able to re~e-ive~-light transmitted from at least a portion of that part of the i~ :
sensor face which receives light from the light emitter.
, ~ .
~. ~
W093/23738 ~` PCT/D~93/00171 In another aspect, the invention relates to a method for ~-inducing and detecting fluorescence in a fluid medium containing at least one fluorophore, the method compri4ing: ~
- transmitting light from a light emitter through a sensor body having internally reflecting wall parts at least capable of reflecting light having a wavelength corresponding to the wavelength of the fluorescence and through a sensor face thereof into the fluid medium, at least part of the light being of a wavelength capable of exciting the fluorophores so as to induce the emission of fluorescence, - receiving, through the sensor face, fluorescence emitted by the excited fluorophores in the fluid medium, and -transmitting at least part of the received fluorescence through the sensor body to a light detector adapted to detect the fluorescence emitted by the fluorophores excited by the exciting light, i at least part of the fluorescence transmitted to the detector being fluorescence received through a part of the sensor face 20 from which }ight from the light emitter is emitted to the fluid medium.
The fluid medium will normally be a liquid, but it may also be a mixture of a liquid and a gas, or it may be a gas with, e.g. suspended particles containing fluorophores.
25 The term "fluorophore" is to be understood in its accepted meaning as a substance which, when excited by absorption of electromagnetic irradiation (light), will decay to a lower - energy state with emission of electromagnetic radiation r having a wavelength longer than the wavelength of the ~- 30 exciting electromagnetic irradiation. Many important fluo-- rescent substances may be excited with Ultra Violet light and will decay with emission of light in the visual range, but ~there are also substances which both absorb and emit light in 2 ~ 3 t i; ? ~
W093~23738 PCT/DK93/00171 the Ultra Violet region, as well as substances which both absorb and emit light in the visual region, and substances which emit in the Near Infra Red-region. The apparatus according to the present invention can be used for measurements in connection with any of these types of substances, although it will be described in the following with particular emphasis on measurements on substances which absorb in the Ultra Violet region and emit visual light.
A central feature of the apparatus of the invention is the sensor body, which constitutes a particularly advantageous functional interface between on the one hand the light emitter and the light detector and on the other hand the fluid medium. One function of the sensor body is to establish such a geometrical position relation between on the one hand the liquid medium and on the other hand the light emitter and the detector that the detector is capable of receiving light from the same part of the liquid medium as has been excited by illumination with the exciting light. The importance of this relation resides in the fact that in a highly turbid medium, the available fluid volume which can be excited is represented by a layer having a thickness of only a few mm, or even only a fraction of a mm, immediately in front of the sensor face and that any part of this layer which has not ``
received the exciting light will not contribute to the active measuring volume.
Thus, in a preferred embodiment, the relative positioning of~
the light emitter, the sensor face, and the optical detéctor ~
is such that light emitted by the light emitter is able to directly illuminate the predominant part of the sensor face.
On the other hand, as the wall parts are capable of`
reflecting the exciting light, any part of the emitted light which is not directly directed to the sensor face-~i-ll~-be transmitted thereto through reflection on the walls.
In another preferred embodiment made possible through the use of the sensor body interface, the relative positioning of the 2 ~ 3~?~
light emitter, the sensor face, and the optical detector is such that the detector is able to directly receive light from a predominant proportion of the illuminated part of the sensor face. Reflection on the walls of the sensor will then further enhance the amount of fluorescence which becomes available for detection.
The exciting light is preferably pulsed. This gives several advantages. Thus, pulsing makes it possible to compensate for any background signal in the measurement. In some of the important types of measurements on fluids containing biological material, e.g., in purification plants, day-light may generate an error signal, which can be compensated for by subtracting the background signaL from the peak fluorescence signal following excitation. Another important advantage of the usè of pulsed induction of fluorescence is that also the fluorescence generated is measured as a pulsed signal, whereby the peak intensity may be orders of magnitude higher 1~`
than when exciting the fluid medium with continuous light having the same mean intensity. Thus, when pulsing the output of the light source, the peak intensity of the light pulses may correspond to the intensity emitted from a continuous 50-~- ~ lOO kW light source.
, When using a pulsed light source, care -should be taken to shield the detecting electronics from noise generated by the light source and its controlling electronics. An advantageous way of reducing the noise due to the pulsed light source is - to separate the pulsing and the detecting electronics physically. This may be accomplished by the use of fibre optics. Thus, in a preferred embodiment of the apparatus according to the invention, the li~ht emitter is a light ~ emitting end of a fibre optical means, such as an optical -~ fibre bundle, having a light receiving end adapted to receive ~- light from a light source.
.
~ The fact that an optical fibre emits light from a very small : ~ ~35 area and at a maximum angle determined by the fibre material -~
2 ~ ~ n ,~
and the wavelength of the light, the maximum angle being smaller for Ultra Violet-transmitting fibres than for fibres transmitting visual light, is compensated for by the fact that the light emitting end of the fibre optical means is positioned at a distance from the sensor face. As will be explained in greater detail in connection with the drawing, this configuration makes it possible for the fibre optical means to illuminate and thus excite a satisfactorily large area of a turbid medium.
It is believed that the special way of using fibre optics - where the light emitting end thereof is kept at a distance from the sensor face, thus enlarging the area of illumination `
and at the same time permitting a detector to "see" all or a considerable proportion of the illuminated area is novel E~E
lS se, and thus, a particular aspect of the present invention can be expressed as an apparatus for inducing and measuring fluorescence in a fluid medium containing at least one fluorophore, the apparatus comprising:
a light emitter adapted to emit light of a wavelength able to excite fluorophores in the fluid medium, - optical fibre means adapted to receive and guide exciting light from the light emitter and having a light emitting end, - a light detector adapted to detect fluorescence emitted by the fluorophores excited by the exciting light, - a sensor body having a sensor face adapted to be exposed to the fluid medium, - the sensor body being adapted to receive exciting-light - ~
; from the light emitting end of the optical fibre means, and~
to transmit the received exciting light into the fluid medium, to receive fluorescence emitted by excited fluorosphores in the fluid medium, and to transmit at~least part of the received fluorescence to the light detector,~
the sensor face being positioned in a distance from-~he-l-ight emitting end of the optical fibre means, and the relative positioning of the light emitting end of the optical fibre means, the sensor face, and the optical detector being such that light emitted by the optical fibre means is able to ~1 3fi .~.~`.?
W093~23738 PCT/DK93/00171 illuminate a part of the sensor face, and such that the detector is able to receive light from at least a part of the thus illuminated part of the sensor face.
Depending on the particular measurement to be performed, the S pulsing may be performed with time intervals varying from fractions of a second to the order of minutes or hours. The duration of each single pulse is normally of the order of fractions of a second.
Alternatively, in a preferred embodiment of the present invention, a measurement may be performed by transmitting a series of pulses, such as 12 to 200 pulses, measuring the generated fluorescence in the peaks and between the peaks and assigning the mean value of the measured fluorescence to be the actual value. The pulses in the measurement may in principle be generated at any frequency; at present, a frequency of in the order of 4 Hz is preferred. Measurements may be performed at regular intervals, such as lO to 60 times or~more an hour. Naturally the frequency of the pulses in the measurement, the number of pulses in the measurement and the 20~ time interval in which the measurements are performed is adapted to each other. If e.g. 180 measurements are performed an hour, the number of pulses used for each measurement is usually in the order of 4-32.
- ~ ;
For the generation of the exciting light, a wide variety of-~ 25 light sources may be used, ranging from Ultra Violet sources to broad-band sources, depending on the properties of the fluorophores to be measured. For most applications, the exciting light will be light in the Ultra Violet region.
~ The wavelength range of the exciting light is normally ~ 3~0 limited, e.g., by incorporating, in the light emitter, light filtering means permitting the light emitter to emit filtered light. In a preferred embodiment, the filtering means is placed between the light source and the light receiving end of the fibre optics.
' ' ,,,,,,...... .. - -- - - -The sensor body is normally a monolithic body, but it is possible, and within the scope of the present invention, to establish the function of the sensor body by combination of `
two or more individual parts which together constitute the S sensor body. The sensor body may be a hollow body, which may present the advantage that the amount of material in the light path which could potentially generate fluorescence (most materials are capable of some degree of fluorescence) is reduced, but for most practical purposes, it is preferred that the sensor body is solid. The use of a solid sensor body has the advantage that a compact and robust design can be obtained, and that the danger of formation of water of '`~
condensation at critical internal parts of the apparatus is obviated or considerably reduced.
The sensor body may be made of any material which is capable of effectively transmitting the exciting light and the fluorescence, such as quartz or sapphire when the exciting light is in the Ultra Violet region,' or glass when the exciting light is in the visual'range. Due to the exciting ~20 light travelling in the sensor body, fluorescence from the material constituting the sensor body may occur, depending on the material of the sensor body. Therefore, it is important to select a~sensor body material which will emit as little fluorescence as possible. For most cases, this means that a high purity of the sensor body material should be secured, as impurities will tend to increase the amount of fluorescence.
It is preferred that the sensor body has a substantially ~~ - '~
symmetrical cross section in a direction transverse to the `' `
' 30 sensor face. In this connection, "transverse" should not be understood as limited to "perpendicular to", but rather --includes both the perpendicular relationship and any slanted or oblique relationship. A suitable method of producing- a`'- -sensor body of the kind disclosed herein is to cut a piece`~of a rod of the material in question, e.g. a quartz rod, and subjecting the piece to working and polishing so as to bring it into a desired shape and establish end faces suitable as ~3 ~ rj I
W093t23738 PCT/DK93/00171 sensor face and face receivlng the detector and the light emitter, respectively.
The cross section of the sensor body may, e.g., be substan-tially elliptical, in particular substantially circular.
S The reflective property of the wall parts of the sensor body are suitably obtained due to a coating of the wall parts. The coating may be any suitable coating resulting in the desired reflecting properties, such as a coating selected from dielectric coatings and metal coatings. The method of application of the coating will be adapted to the coating material and may comprise application from a liquid or from a vapour. In a presently preferred embodiment, the sensor body is coated with an external coating with aluminum applied by vapour deposition.
lS The sensor body will normally be an elongated body.
The dimensions of the sensor body may vary over a wide range.
Normally, the sensor body will have a length in the range of
3-5Q mm, preferably in the range of 5-50 mm, more preferably in the range of 10-25 mm, such as about lS mm. The diameter 1 20 is normally in the range of 2-40 mm, preferably in the range of 3-30 mm, more preferably in the range of 5-20 mm, such as about lO mm. I
:~ ~
The practical physical appearance of the apparatus according to the invention can be varied in many ways, depending on the environment in which the apparatus is to be used. As an example may be mentioned an embodiment wherein at least a portion of the sensor body is adapted to extend into the fluid; in this embodiment, the sensor face is less prone to become covered or fouled by, e.g. microorganisms or any material present in the fluid to be processed.
~:
As will be understood from the above discussion, the light emitter may be, and often preferably is, the light emitting : - .
end of a fibre optical means, but it is also within the scope of the invention that a light source arranged in connection with the sensor body serves as the-light emitter. --The invention will now be described with reference to the5 drawing where:
- Fig. l is a schematic figure of a presently preferred embodiment of a sensor according to the invention, - Fig. 2 is a detailed drawing of the operation of the sensor body, and - Fig. 3 shows a test performed to illustrate the increased sensitivity of a sensor according to the present invention compared to a commercially available sensor.
- Fig. 4 illustrates a comparison of the sensitivity of the sensor according to the invention and the sensitivity of the commercially available sensor; the sensitivity is taken as the sensor output as a function of the distance from a fluorescent object to the end of the sensor.
In~Fig. l, light from an exciting light source lO, such as W
light from a flash lamp controlled by electronia controlling means (not shown), is collimated by a lens 12. From the collimated light, a band pass filter 14 removes substantially all light not having a wavelength able to excite the fluorophores in the-fluid. After filtration, the light passes a lens 16 and is thereby focused onto a light receiving~end il - 2~5 21 of a fibre optical means 20, preferably a fiber bundle known ~er se. The exciting light is then guided by the fibre-~ --optical means 20 to a light emitting end 22 of the fibre ~
optical means 20 and from there to a solid sensor body 30 of a material which is capable of transmitting the excitlng light and fluorescence generated as a result of excitation with the exciting light, such as quartz. In Fig~ l, the~body is shaped as a circular cylinder having end parts 3~ an~--32 and a wall part 33 constituted by the outer cylindrical~`~
circumference and comprising a vapour-deposited aluminum coating of a thickness of a few ~m, constituting a reflecting layer surrounding the sensor body 30. The exciting light then :
2 ~
travels through the sensor body 30 to the end part 32 which constitutes the sensor face of the body adapted to be in contact with the fluid containing the fluorophores.
~ ~ :
; When the exciting light enters the sensor body 30, a major 5 part~thereof illuminates the sensor faae 32, while a part of ~this~;light~will be directed toward the~ wall part 33 of the sensor~body 30. The part of~the exciting light directed ,, toward~this~wall part 33 will be reflected by~the wall part 33 wherèby~the~re~flected light~will be directed to the sensor lO~ face~32~,~thus minlmi~zing~light losses~
,When~the~sensor~face~32~ls~exposed to a fluid~containing f`luorophores~exc~itab1e~by~the~;exciting light, fluorescence will~be~ generated in the fluid when the exc~itingllight enters "`è
~ the~flui~d.~Part of this fluorescence will be directed toward ;',,~ -,l5~the~sensor~face~32~-`and~will~enter-~the sensor body 30.
'A ~ ~detector~4a~is~pQsitioned~in a~pos~ition at the end p ~ '~3~ ~ e~it~`can~directly detect fluorescence from the ma~or part~o~the~sensor~face 3~2~. In~addition~, the detector ,, 40~reeeives~f1uorescence~reflected from the~wall parts 33, 0- ~ reby ~a~larger amount~o~f~the~fluoresc~ence from the excited ~or~phores~;~is~detected.:~Th`ere~for~e,~the~reflecting wall ~ ï
pa ~ -~33~increase~the sensitivity~of ~he sensor.
Thè-~detector~40~;transforms~the~light~s~ignal received into an eIec,tr~ical~signal~rep~esentat ~ ~:of-~the amount~f light ;'' ~-25~ received.~The~electrical~signal~is~conducted to recording and/or~calculating and/or process-regulating electronics, not shown.
When the f1uid mediu~ to~which the sensor face~32 is exposed ~;
3,0 ~ is~a~highly turbid~medium,~such as sewage or wa$te water treatèd with a mixéd~culture~of microorganisms in a water purification~plant,;~the~exc~iting light~wi~ll not travel more than~a few mm~into~the~;medium before it is~ absorbed to a very considerable~extent,~ and likewise, the fluorescence generated 2:~3fi ~0.`~ . -due to the excitation will not be detectable through a medium thickness of more than very few mm. For this reason, it is important, in order to obtain a realrstiç volume on which to perform the measurement, that the sensor face has a relative large area through which a reasonable volume of the medium can be exposed to the exciting light, and it is also important that the detector is able to receive the fluorescence from at least a portion of the area where the illumination takes~place, preferably from a predominant lO~ portion o~f this area.~Fig.~ 2 illustrates these cons~iderations.
~ . ~
In Fig.~ 2~, a~light emitter~in the form of a light emitting end~22~of~a~ fibre~optical méans 20;is~seen abutting the sensor body 30. As described fùrther above, light emitted from~fibre optical means is emitted having a maximum angle -defined~by~the~materlals`dèfining~the core andithe cladding of~the~individùal~fibres~ Lines I and II illustrate this màx~ um~angle~for~a~prèfe`rred~embodiment in which the fibre Z~O~optical~means 20~is~à~fibre~opt~icàl bundle. It~ is seen that part~of~the~emitted~excit1ng~light (e~.g. II) is~directed ; toward~the~wall~ parts ~l3 and,~after a ref~lection on the wall parts 33,~;iæ`~directed~toward the~sensor face 32~. It is seen that ln this preferred~embodimènt, the~predominant part of 25 ~ the~senso~fàce 32~is;~illuminated~d~irectly by the exciting light~ In~this preferred~embodiment, the fibre optical bundle 20~is~positioned~so~as~to primàrily i}luminate the part of ;the ~sensor-~face 32~which is clos-st to the detector 4Q.~
Lines III and IV in Fig. 2 give examples of fluorescence ~30 ~generated by the`flùorophorès which either directly (III) or through a reflection on the wall parts 33 (~V) is directed-to the ensitiv- surface 50 of~the detector. It is evident that ~ the~reflection at~the~wall parts 33 considerably incre~ses-s~ the amount of the fluorescence received by the detector`40.
J: : ~
23 ~fi .~i~
EXAMPLE
As an example, a preferred embodiment of a sensor according to the present invention may comprise:
` .
light emitter }0 bulb type xenon flash lamp S collimating lens 12 aspheric condenser lens filter 14 W band pass filter 300-400 nm focusing lens 16 plano convex quartz lens fiber~ optical bundle 20 quartz/guartz fibres, 3 mm diameter 10 sensor body 30 quartz cylinder reflecting coating ; aluminum, vapour deposited ;~ ` filter 42 visual band pass filter 400-500 nm detector 40 PIN silicon diode ::
~The~sensor`body is~preferably a solid quartz rod having dimensions:
diameter : l0 mm length : l5 _.
Of~ course, the~shape and size~of the~sensor body may be 20~ different.~As~the~functian of the body ~s solely to guide the light,~any~shàpe, size,` ànd~materials sùpporting this purpose may be used~
The ~exterlor;of th-~quartz body~has preferably been polished whereafter the wall parts have been coated with a few ~m of aluminum applied by vapour deposition.
The lens-s;are preferably selected so that the light focused onto~an~end of the fibre optical bundle has a shape and size substantially identical to those of the light emitting parts of the light source.
;''.` :; :
:;:
, Ç~- A ~
As the sensor body is polished, it is sufficient to position the light emitting end of the fibre optical bundle outside the sensor body, preferably in abutting relationship thereto as shown in Fig. 2, so that the exciting light, when exiting the fibre optical bundle, enters the sensor body and illuminates the sensor face as described above. It will be understood, however, that it is also possible - and may be preferred for some purposes - to design the sensor body in a shape which secures a better transfer of the light from the fibre optical bundle to the sensor body, such as, e.g., with a bore receiving the fibre optical bundle. ~`
The light source of the sensor is preferably pulsed with a frequency of 4 Hz during the measurements. The detector then generates an electrical signal corresponding to the pulses of ^
fluorescence generated by the fluorophores. Any signal corresponding to a background signal e.~. daylight, is measured between the fluorescence pulses. It is preferred that the individual peaks from the detected fluorescence ¦
peaks are converted to a mean value for the measurement. The ; 20 background signal is also preferred to be a mean value of the detected background signal during the measurement.
For the detector to be able to precisely detect the fluo-rescence pulses, the sensitive area of the detector should preferably be relatively small, such as an area of about 5-10-mm2, or smaller. In the above-mentioned presently preferred embodiment, the sensitive area of the detector is about ?-~mm2. The size of the sensitive area of the deteator defines -- - ;
the speed of the detector. Pulses having a small duration in ~
time should be detected by a detector having a small sensitive area.
Of course a smaller sensitive area is less sensitive than--a-- larger sensitive area. However, this may be compensated ~or by strong pulses of the exciting light generating more fluorescence than if the light was continuous with the same 3S mean intensity. Furthermore, a detector having a large ;." 1 ~
sensitive area generates more noise than a detector having a small sensitive area.
This also has the advantage that detectors with small sensitive areas are usually cheaper than detectors having large sensitive areas. For this sensor, a small commercially available silicon PIN photodiode is suitable.
When combining the detector having a small sensitive area and - the pulsed mode of the light source, an overall larger output signal may be obtained without reducing the signal to noise ratio below that obtained by using a sensor operating in continuous mode and using a detector having a larger sensitive area. The signal to noise ratio depends on both the noise generated by the sensitive area of the detector and the amplification of the signal.
, When subtracting the background signal from the peak of the fluorescence pulse, the amount of fluorophores in the fluid may be evaluated or, if desired, calculated on the basis of suitable calibration in manners known E~E se.
: i ,.
COMPARISON EXPERIMENT
~ 20 To illustrate the increased sensitivity of a sensor according -~ to the present invention compared to the commercially available sensor, a test has been performed in the aeration , tank of the Central Purification Plant in the city of Viborg, Denmark.
The aeration tank of the purification plant contains a mixed culture of microorganisms. The microorganisms in the mixed ;~
culture contain NADH which is a fluorophore. The amount of NADH in the microorganisms depend on the activity level of the organisms.
.
, 2 ~ n, ~
W093/23738 PC~/DK93/00171 In the test, a sensor accord1ng to the present invention and corresponding to the preferred embodiment il~ustrated herein and described in the Example above was-tested against a--commercially availa~le fluorescence sensor available from BioChem Technology Inc., Malvern, Pennsylvania, U S.A.
.
The sensor according to the invention was operated with one measurement per minute, 200 pulses per measurement at 4 Hz.
The commerciaI sensor comprises a front part adapted to be exposed to a liquid environment in which a fluorophore is to be determined. The front part comprises a circular channel through which a light beam from a continuous lamp travels to and through a front window which is sealingly mounted at the front end of the channel. The channel is surrounded by an annular light detector positioned behind annular optical filters and positioned as close to the front end of the sensor as possible, taking the optical filters into account.
Due~to this design, the detector is not able to receive fluorescent light through the part of the window which transmits light from the light emitter, and the "field of view" of the detector therefore has a "blind spot"
immediately in front of the window, as the field of view does not comprise any substantial part of the volume of the liquid environment which is immediately adjacent to the window.
.
Both sensors were tested during the same period of time, and they were p}aced in the same measuring site in the fluid. The - test was performed over 16 hours. ~- -.: , Fig. 3 shows the current output from the two detectors, measured in mA, as a function of time, measured in hours.
Fig. 3A shows the output from a sensor according to the ` r present invention, and Fig. 3B shows the output from the- - - -commercially available sensor.
As is seen from Fig. 3, the output from the sensor according - to the present invention is a factor 2.5 higher than that of .
:
2~ 2~9 the commercially available sensor. The higher output is generated by the sensor according to the invention operating in pulsing mode as the signal to noise ratio of this æensor ` ~ makes this amplification possible.
~'~ ; 5 ~ But what is more important, the sénsor accordinq to the present invention ha~s~a~dynamic range (the currènt difference between~the~highest~and the~1Owest current in~the measurement)~of~3.Z mA,~whereas the commercially avaiIable sensor~has~a~dynamic ra~nge of only 0.2 mA
10~ From~this~;it~is~evident~that the sensor~according to the present'~invention~is~extremely~useful for~when performing exact~and~highly~sensitive~meaæuremènts for~e~.g. precisel~ i `
monitoring`a~process in~mèdium of high~`turbidity such as a `;
purifica~t`ion plant. ~ '"' Fig~ 4~il1ustrates~t e sensitiv ~of~the sensor according to the~invent~i~on,~operated~ in~the~samé~;mànner`as~above, compared ;to~the'~co~merc~iàlly~available~sensor~`. The~sensitivity was ' 1' d ~ ~ d~by~pos1tion}ng'~a flùoreæcent objeCt, in the form~
of~ a~piçce~of~paper,~ in~front of~th~senæors while measuring 20~`the~séns~or outpUt~as~a~runction of~the~di~tancè~'~from the end ' - - ~ ' of the sensor to the paper. The~-maximum output~of the sensor :~' according;to~the invention~is regulated~`to }; the output of th~i ~ ialiy~available sensor ~is~ regulated~in relation ~i' 25~ this~f ~ re~ ;the~consegùence~of the-blind~spot of the commercia11y~'available~sensor~is~obvious: this`~sensor has maximum;'~sensitivity~when~the~fluorescing object is at a distance of~3 mm from~the sensor. The~sensor according to the i~v ntion~has~optlmùm sensitivlty~wh-n the~fluorQscent medium ~I
ls~as close to th-~;end~of~thè~s-nsor as~possibl~. (' In~a~turbid~medlum,~such~as ln a~waste water purificatlon plant,~light~i's almost~tota;lly~attenuated;~after~-trave1llng ' only~a few mm~in~the~medium.~Thus, the~major part of the y W093/23738 PCT/DK93/00171 f, generated fluorescence is generated just in front of the front end of the sensor and, of course, in the area of the front end of the sensor in which the medium is illuminate~:
in the blind spot of the commercially available sensor. Thus, it is obvious that the optimum sensor for use in turbid media should have optimum sensitivity as close to the end of the sensor as possible as well as a large area in which fluorescence is generated and from which this fluorescence may be detected.
The optimum sensitivity of the commercially available sensor is 20% of that of the sensor according to the invention. This is again caused by the sensor according to the present invention offering a better signal due to the better signal to noise ratio offered by the use of pulsed mode operation and a more suitable structure of the sensor body; the reflecting coating on the sensor body also plays a role in - increasing the sensitivity of the sensor according to the invention.
- Even though the sensor according to the present invention, thus, possesses optimum features and sensitivity for use in the generation and detection of fluorescence in this type of medium, the sensor possesses the same features in less turbid ~' media where the exciting light is launched a larger distance into the medium. ~ -The sensor according to the invention offers the same degree- - - -of precise determinations in less turbid medià as the overlap ^ -- -of the volume of the medium excited by the exciting light and ~`~~
the volume from which emitted fluorescence may be detected is large throughout the medium.
.
:~ ~
The practical physical appearance of the apparatus according to the invention can be varied in many ways, depending on the environment in which the apparatus is to be used. As an example may be mentioned an embodiment wherein at least a portion of the sensor body is adapted to extend into the fluid; in this embodiment, the sensor face is less prone to become covered or fouled by, e.g. microorganisms or any material present in the fluid to be processed.
~:
As will be understood from the above discussion, the light emitter may be, and often preferably is, the light emitting : - .
end of a fibre optical means, but it is also within the scope of the invention that a light source arranged in connection with the sensor body serves as the-light emitter. --The invention will now be described with reference to the5 drawing where:
- Fig. l is a schematic figure of a presently preferred embodiment of a sensor according to the invention, - Fig. 2 is a detailed drawing of the operation of the sensor body, and - Fig. 3 shows a test performed to illustrate the increased sensitivity of a sensor according to the present invention compared to a commercially available sensor.
- Fig. 4 illustrates a comparison of the sensitivity of the sensor according to the invention and the sensitivity of the commercially available sensor; the sensitivity is taken as the sensor output as a function of the distance from a fluorescent object to the end of the sensor.
In~Fig. l, light from an exciting light source lO, such as W
light from a flash lamp controlled by electronia controlling means (not shown), is collimated by a lens 12. From the collimated light, a band pass filter 14 removes substantially all light not having a wavelength able to excite the fluorophores in the-fluid. After filtration, the light passes a lens 16 and is thereby focused onto a light receiving~end il - 2~5 21 of a fibre optical means 20, preferably a fiber bundle known ~er se. The exciting light is then guided by the fibre-~ --optical means 20 to a light emitting end 22 of the fibre ~
optical means 20 and from there to a solid sensor body 30 of a material which is capable of transmitting the excitlng light and fluorescence generated as a result of excitation with the exciting light, such as quartz. In Fig~ l, the~body is shaped as a circular cylinder having end parts 3~ an~--32 and a wall part 33 constituted by the outer cylindrical~`~
circumference and comprising a vapour-deposited aluminum coating of a thickness of a few ~m, constituting a reflecting layer surrounding the sensor body 30. The exciting light then :
2 ~
travels through the sensor body 30 to the end part 32 which constitutes the sensor face of the body adapted to be in contact with the fluid containing the fluorophores.
~ ~ :
; When the exciting light enters the sensor body 30, a major 5 part~thereof illuminates the sensor faae 32, while a part of ~this~;light~will be directed toward the~ wall part 33 of the sensor~body 30. The part of~the exciting light directed ,, toward~this~wall part 33 will be reflected by~the wall part 33 wherèby~the~re~flected light~will be directed to the sensor lO~ face~32~,~thus minlmi~zing~light losses~
,When~the~sensor~face~32~ls~exposed to a fluid~containing f`luorophores~exc~itab1e~by~the~;exciting light, fluorescence will~be~ generated in the fluid when the exc~itingllight enters "`è
~ the~flui~d.~Part of this fluorescence will be directed toward ;',,~ -,l5~the~sensor~face~32~-`and~will~enter-~the sensor body 30.
'A ~ ~detector~4a~is~pQsitioned~in a~pos~ition at the end p ~ '~3~ ~ e~it~`can~directly detect fluorescence from the ma~or part~o~the~sensor~face 3~2~. In~addition~, the detector ,, 40~reeeives~f1uorescence~reflected from the~wall parts 33, 0- ~ reby ~a~larger amount~o~f~the~fluoresc~ence from the excited ~or~phores~;~is~detected.:~Th`ere~for~e,~the~reflecting wall ~ ï
pa ~ -~33~increase~the sensitivity~of ~he sensor.
Thè-~detector~40~;transforms~the~light~s~ignal received into an eIec,tr~ical~signal~rep~esentat ~ ~:of-~the amount~f light ;'' ~-25~ received.~The~electrical~signal~is~conducted to recording and/or~calculating and/or process-regulating electronics, not shown.
When the f1uid mediu~ to~which the sensor face~32 is exposed ~;
3,0 ~ is~a~highly turbid~medium,~such as sewage or wa$te water treatèd with a mixéd~culture~of microorganisms in a water purification~plant,;~the~exc~iting light~wi~ll not travel more than~a few mm~into~the~;medium before it is~ absorbed to a very considerable~extent,~ and likewise, the fluorescence generated 2:~3fi ~0.`~ . -due to the excitation will not be detectable through a medium thickness of more than very few mm. For this reason, it is important, in order to obtain a realrstiç volume on which to perform the measurement, that the sensor face has a relative large area through which a reasonable volume of the medium can be exposed to the exciting light, and it is also important that the detector is able to receive the fluorescence from at least a portion of the area where the illumination takes~place, preferably from a predominant lO~ portion o~f this area.~Fig.~ 2 illustrates these cons~iderations.
~ . ~
In Fig.~ 2~, a~light emitter~in the form of a light emitting end~22~of~a~ fibre~optical méans 20;is~seen abutting the sensor body 30. As described fùrther above, light emitted from~fibre optical means is emitted having a maximum angle -defined~by~the~materlals`dèfining~the core andithe cladding of~the~individùal~fibres~ Lines I and II illustrate this màx~ um~angle~for~a~prèfe`rred~embodiment in which the fibre Z~O~optical~means 20~is~à~fibre~opt~icàl bundle. It~ is seen that part~of~the~emitted~excit1ng~light (e~.g. II) is~directed ; toward~the~wall~ parts ~l3 and,~after a ref~lection on the wall parts 33,~;iæ`~directed~toward the~sensor face 32~. It is seen that ln this preferred~embodimènt, the~predominant part of 25 ~ the~senso~fàce 32~is;~illuminated~d~irectly by the exciting light~ In~this preferred~embodiment, the fibre optical bundle 20~is~positioned~so~as~to primàrily i}luminate the part of ;the ~sensor-~face 32~which is clos-st to the detector 4Q.~
Lines III and IV in Fig. 2 give examples of fluorescence ~30 ~generated by the`flùorophorès which either directly (III) or through a reflection on the wall parts 33 (~V) is directed-to the ensitiv- surface 50 of~the detector. It is evident that ~ the~reflection at~the~wall parts 33 considerably incre~ses-s~ the amount of the fluorescence received by the detector`40.
J: : ~
23 ~fi .~i~
EXAMPLE
As an example, a preferred embodiment of a sensor according to the present invention may comprise:
` .
light emitter }0 bulb type xenon flash lamp S collimating lens 12 aspheric condenser lens filter 14 W band pass filter 300-400 nm focusing lens 16 plano convex quartz lens fiber~ optical bundle 20 quartz/guartz fibres, 3 mm diameter 10 sensor body 30 quartz cylinder reflecting coating ; aluminum, vapour deposited ;~ ` filter 42 visual band pass filter 400-500 nm detector 40 PIN silicon diode ::
~The~sensor`body is~preferably a solid quartz rod having dimensions:
diameter : l0 mm length : l5 _.
Of~ course, the~shape and size~of the~sensor body may be 20~ different.~As~the~functian of the body ~s solely to guide the light,~any~shàpe, size,` ànd~materials sùpporting this purpose may be used~
The ~exterlor;of th-~quartz body~has preferably been polished whereafter the wall parts have been coated with a few ~m of aluminum applied by vapour deposition.
The lens-s;are preferably selected so that the light focused onto~an~end of the fibre optical bundle has a shape and size substantially identical to those of the light emitting parts of the light source.
;''.` :; :
:;:
, Ç~- A ~
As the sensor body is polished, it is sufficient to position the light emitting end of the fibre optical bundle outside the sensor body, preferably in abutting relationship thereto as shown in Fig. 2, so that the exciting light, when exiting the fibre optical bundle, enters the sensor body and illuminates the sensor face as described above. It will be understood, however, that it is also possible - and may be preferred for some purposes - to design the sensor body in a shape which secures a better transfer of the light from the fibre optical bundle to the sensor body, such as, e.g., with a bore receiving the fibre optical bundle. ~`
The light source of the sensor is preferably pulsed with a frequency of 4 Hz during the measurements. The detector then generates an electrical signal corresponding to the pulses of ^
fluorescence generated by the fluorophores. Any signal corresponding to a background signal e.~. daylight, is measured between the fluorescence pulses. It is preferred that the individual peaks from the detected fluorescence ¦
peaks are converted to a mean value for the measurement. The ; 20 background signal is also preferred to be a mean value of the detected background signal during the measurement.
For the detector to be able to precisely detect the fluo-rescence pulses, the sensitive area of the detector should preferably be relatively small, such as an area of about 5-10-mm2, or smaller. In the above-mentioned presently preferred embodiment, the sensitive area of the detector is about ?-~mm2. The size of the sensitive area of the deteator defines -- - ;
the speed of the detector. Pulses having a small duration in ~
time should be detected by a detector having a small sensitive area.
Of course a smaller sensitive area is less sensitive than--a-- larger sensitive area. However, this may be compensated ~or by strong pulses of the exciting light generating more fluorescence than if the light was continuous with the same 3S mean intensity. Furthermore, a detector having a large ;." 1 ~
sensitive area generates more noise than a detector having a small sensitive area.
This also has the advantage that detectors with small sensitive areas are usually cheaper than detectors having large sensitive areas. For this sensor, a small commercially available silicon PIN photodiode is suitable.
When combining the detector having a small sensitive area and - the pulsed mode of the light source, an overall larger output signal may be obtained without reducing the signal to noise ratio below that obtained by using a sensor operating in continuous mode and using a detector having a larger sensitive area. The signal to noise ratio depends on both the noise generated by the sensitive area of the detector and the amplification of the signal.
, When subtracting the background signal from the peak of the fluorescence pulse, the amount of fluorophores in the fluid may be evaluated or, if desired, calculated on the basis of suitable calibration in manners known E~E se.
: i ,.
COMPARISON EXPERIMENT
~ 20 To illustrate the increased sensitivity of a sensor according -~ to the present invention compared to the commercially available sensor, a test has been performed in the aeration , tank of the Central Purification Plant in the city of Viborg, Denmark.
The aeration tank of the purification plant contains a mixed culture of microorganisms. The microorganisms in the mixed ;~
culture contain NADH which is a fluorophore. The amount of NADH in the microorganisms depend on the activity level of the organisms.
.
, 2 ~ n, ~
W093/23738 PC~/DK93/00171 In the test, a sensor accord1ng to the present invention and corresponding to the preferred embodiment il~ustrated herein and described in the Example above was-tested against a--commercially availa~le fluorescence sensor available from BioChem Technology Inc., Malvern, Pennsylvania, U S.A.
.
The sensor according to the invention was operated with one measurement per minute, 200 pulses per measurement at 4 Hz.
The commerciaI sensor comprises a front part adapted to be exposed to a liquid environment in which a fluorophore is to be determined. The front part comprises a circular channel through which a light beam from a continuous lamp travels to and through a front window which is sealingly mounted at the front end of the channel. The channel is surrounded by an annular light detector positioned behind annular optical filters and positioned as close to the front end of the sensor as possible, taking the optical filters into account.
Due~to this design, the detector is not able to receive fluorescent light through the part of the window which transmits light from the light emitter, and the "field of view" of the detector therefore has a "blind spot"
immediately in front of the window, as the field of view does not comprise any substantial part of the volume of the liquid environment which is immediately adjacent to the window.
.
Both sensors were tested during the same period of time, and they were p}aced in the same measuring site in the fluid. The - test was performed over 16 hours. ~- -.: , Fig. 3 shows the current output from the two detectors, measured in mA, as a function of time, measured in hours.
Fig. 3A shows the output from a sensor according to the ` r present invention, and Fig. 3B shows the output from the- - - -commercially available sensor.
As is seen from Fig. 3, the output from the sensor according - to the present invention is a factor 2.5 higher than that of .
:
2~ 2~9 the commercially available sensor. The higher output is generated by the sensor according to the invention operating in pulsing mode as the signal to noise ratio of this æensor ` ~ makes this amplification possible.
~'~ ; 5 ~ But what is more important, the sénsor accordinq to the present invention ha~s~a~dynamic range (the currènt difference between~the~highest~and the~1Owest current in~the measurement)~of~3.Z mA,~whereas the commercially avaiIable sensor~has~a~dynamic ra~nge of only 0.2 mA
10~ From~this~;it~is~evident~that the sensor~according to the present'~invention~is~extremely~useful for~when performing exact~and~highly~sensitive~meaæuremènts for~e~.g. precisel~ i `
monitoring`a~process in~mèdium of high~`turbidity such as a `;
purifica~t`ion plant. ~ '"' Fig~ 4~il1ustrates~t e sensitiv ~of~the sensor according to the~invent~i~on,~operated~ in~the~samé~;mànner`as~above, compared ;to~the'~co~merc~iàlly~available~sensor~`. The~sensitivity was ' 1' d ~ ~ d~by~pos1tion}ng'~a flùoreæcent objeCt, in the form~
of~ a~piçce~of~paper,~ in~front of~th~senæors while measuring 20~`the~séns~or outpUt~as~a~runction of~the~di~tancè~'~from the end ' - - ~ ' of the sensor to the paper. The~-maximum output~of the sensor :~' according;to~the invention~is regulated~`to }; the output of th~i ~ ialiy~available sensor ~is~ regulated~in relation ~i' 25~ this~f ~ re~ ;the~consegùence~of the-blind~spot of the commercia11y~'available~sensor~is~obvious: this`~sensor has maximum;'~sensitivity~when~the~fluorescing object is at a distance of~3 mm from~the sensor. The~sensor according to the i~v ntion~has~optlmùm sensitivlty~wh-n the~fluorQscent medium ~I
ls~as close to th-~;end~of~thè~s-nsor as~possibl~. (' In~a~turbid~medlum,~such~as ln a~waste water purificatlon plant,~light~i's almost~tota;lly~attenuated;~after~-trave1llng ' only~a few mm~in~the~medium.~Thus, the~major part of the y W093/23738 PCT/DK93/00171 f, generated fluorescence is generated just in front of the front end of the sensor and, of course, in the area of the front end of the sensor in which the medium is illuminate~:
in the blind spot of the commercially available sensor. Thus, it is obvious that the optimum sensor for use in turbid media should have optimum sensitivity as close to the end of the sensor as possible as well as a large area in which fluorescence is generated and from which this fluorescence may be detected.
The optimum sensitivity of the commercially available sensor is 20% of that of the sensor according to the invention. This is again caused by the sensor according to the present invention offering a better signal due to the better signal to noise ratio offered by the use of pulsed mode operation and a more suitable structure of the sensor body; the reflecting coating on the sensor body also plays a role in - increasing the sensitivity of the sensor according to the invention.
- Even though the sensor according to the present invention, thus, possesses optimum features and sensitivity for use in the generation and detection of fluorescence in this type of medium, the sensor possesses the same features in less turbid ~' media where the exciting light is launched a larger distance into the medium. ~ -The sensor according to the invention offers the same degree- - - -of precise determinations in less turbid medià as the overlap ^ -- -of the volume of the medium excited by the exciting light and ~`~~
the volume from which emitted fluorescence may be detected is large throughout the medium.
.
Claims (23)
1. An apparatus for inducing and detecting fluorescence in a fluid medium containing at least one fluorophore, the apparatus comprising:
- a light emitter (10, 22) adapted to emit light of a wavelength capable of exciting fluorophores in the fluid medium, - a light detector (40) adapted to detect fluorescence emitted by the fluorophores excited by the exciting light, characterized in that the apparatus further comprises - a solid sensor body (30) having internally reflecting wall parts (33) capable of reflecting at least light having a wavelength corresponding to the wavelength of the fluorescence and having a sensor face (32) adapted to be exposed to the fluid medium, the sensor body (30) being adapted to receive exciting light from the light emitter (10, 22), and to transmit the received exciting light into the fluid medium through the sensor face (32), to receive, through the sensor face (32), fluorescence emitted by excited fluorsphores in the fluid medium, and to transmit at least part of the received fluorescence to the light detector (40), the sensor body (30) being made of a material which is capable of effectively transmitting the exciting light and the fluorescence, both the light emitter (10, 22) and the detector (40) being positioned at positions at the end (31) of the sensor body (30) and in a distance from the sensor face (32), and the relative positioning of the light emitter (10, 22), the optical detector, and the sensor face (32) being such that the detector is able to receive light transmitted from at least a portion of that part of the sensor face (32) which receives light from the light emitter (10, 22).
- a light emitter (10, 22) adapted to emit light of a wavelength capable of exciting fluorophores in the fluid medium, - a light detector (40) adapted to detect fluorescence emitted by the fluorophores excited by the exciting light, characterized in that the apparatus further comprises - a solid sensor body (30) having internally reflecting wall parts (33) capable of reflecting at least light having a wavelength corresponding to the wavelength of the fluorescence and having a sensor face (32) adapted to be exposed to the fluid medium, the sensor body (30) being adapted to receive exciting light from the light emitter (10, 22), and to transmit the received exciting light into the fluid medium through the sensor face (32), to receive, through the sensor face (32), fluorescence emitted by excited fluorsphores in the fluid medium, and to transmit at least part of the received fluorescence to the light detector (40), the sensor body (30) being made of a material which is capable of effectively transmitting the exciting light and the fluorescence, both the light emitter (10, 22) and the detector (40) being positioned at positions at the end (31) of the sensor body (30) and in a distance from the sensor face (32), and the relative positioning of the light emitter (10, 22), the optical detector, and the sensor face (32) being such that the detector is able to receive light transmitted from at least a portion of that part of the sensor face (32) which receives light from the light emitter (10, 22).
2. An apparatus according to claim 1, wherein the relative positioning of the light emitter (10, 22), the sensor face (32), and the optical detector is such that light emitter by the light emitter (10, 22) is able to directly illuminate the predominant part of the sensor face (32).
3. An apparatus according to claim 2, wherein the relative positioning of the light emitter (10, 22), the sensor face (32), and the optical detector is such that the detector is able to directly receive light from a predominant proportion of the illuminated part of the sensor face (32).
4. An apparatus according to any of claims 1-3, wherein the light emitter (10, 22) is a light emitting end of a fibre optical means having a light receiving end adapted to receive light from a light source.
5. An apparatus according to claim 4, wherein the fibre optical means is an optical fibre bundle.
6. An apparatus according to any of the preceding claims, wherein the light emitter (10, 22) is capable of emitting pulsed light.
7. An apparatus according to any of the preceding claims, wherein at least part of the light which the light emitter (10, 22) is capable of emitting is in the Ultra Violet region.
8. An apparatus according to any of the preceding claims, further comprising light filtering means permitting the light emitter (10, 22) to emit filtered light.
9. An apparatus according to any of the preceding claims, wherein the sensor body (30) is a monolithic body.
10. An apparatus according to any of the preceding claims, wherein the sensor body (30) has a substantially symmetrical cross section in a direction transverse to the sensor face (32).
11. An apparatus according to claim 10, wherein the cross section is substantially elliptical.
12. An apparatus according to claim 11, wherein the cross section is substantially circular.
13. An apparatus according to any of the preceding claims, wherein the sensor body (30) is an elongated body.
14. An apparatus according to any of the preceding claims, wherein the sensor body (30) is made of quartz.
15. An apparatus according to any of the preceding claims, wherein the reflective property of the wall parts (33) of the sensor body (30) are due to a coating of the wall parts.
16. An apparatus-according to claim 15, wherein the coating is selected from dielectric coatings and metal coatings.
17. An apparatus according to claim 15 or 16, wherein the coating is ah aluminum coating,
18. An apparatus according to any of the preceding claims, wherein the sensor body (30) has a length in the range of 3-50 mm, preferably in the range of 5-50 mm, more preferably in the range of 10-25 mm, such as about 15 mm.
19. An apparatus according to any of the preceding claims, wherein the sensor body (30) has a diameter of in the range of 2-40 mm, preferably in the range of 3-30 mm, more pre-ferably in the range of 5-20 mm, such as about 10 mm.
20. An apparatus according to any of the preceding claims, further comprising filter means filtering the fluorescence to be detected by the optical detector so that substantially only light having a wavelength in the wavelength region of the fluorescence emitted from the excited fluorophores is received by the detector.
21. A method for including and detecting fluorescence in a fluid medium containing at least one fluorophore, characterized in that the method comprises - transmitting light from a light emitter (10, 22) positioned in a position at the end (31) of a solid sensor body (30) having internally reflecting wall parts (33) capable of at least reflecting light having a wavelength corresponding to the wavelength of the sensor face (32) thereof into the fluid medium, at least part of the light being of a wavelength capable of exciting the fluorophores so as to induce the emission of fluorescence, the sensor body (30) being made of a material which is capable of effectively transmitting the exciting light and the fluorescence, - receiving, through the sensor face (32), fluorescence emitted by the excited fluorophores in the fluid medium, and transmitting at least part of the received fluorescence through the sensor body (30) to a light detector (40) adapted to detect the fluorescence emitted by the fluorophores excited by the exciting light and positioned at a position at the end (31) of the sensor body (30), at least part of the fluorescence transmitted to the detector being fluorescence received through a part of the sensor face (32) from which light from the light emitter (10, 22) is emitted to the fluid medium.
22. A method according to claim 21, wherein the fluid medium is a turbid medium.
23. A method according to claim 21 or 22, wherein the fluid medium is waste water which is being biodegraded by a mixed culture of microorganisms, the microorganisms containing at least one biogenic fluorophore the fluorescence of which is inducible by the exciting light and detectable by the light detector (40).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK92669A DK66992D0 (en) | 1992-05-21 | 1992-05-21 | SENSOR |
| DK0669/92 | 1992-05-21 |
Publications (1)
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|---|---|
| CA2136209A1 true CA2136209A1 (en) | 1993-11-25 |
Family
ID=8096184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002136209A Abandoned CA2136209A1 (en) | 1992-05-21 | 1993-05-19 | Apparatus and method for inducing and detecting fluorescence |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US5557415A (en) |
| EP (1) | EP0641431B1 (en) |
| JP (1) | JPH08504033A (en) |
| AT (1) | ATE157771T1 (en) |
| CA (1) | CA2136209A1 (en) |
| DE (1) | DE69313633T2 (en) |
| DK (2) | DK66992D0 (en) |
| ES (1) | ES2109501T3 (en) |
| NO (1) | NO944427L (en) |
| WO (1) | WO1993023738A1 (en) |
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| US6914250B2 (en) | 1997-03-07 | 2005-07-05 | Clare Chemical Research, Inc. | Fluorometric detection using visible light |
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-
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- 1993-05-19 US US08/343,511 patent/US5557415A/en not_active Expired - Lifetime
- 1993-05-19 CA CA002136209A patent/CA2136209A1/en not_active Abandoned
- 1993-05-19 JP JP5519797A patent/JPH08504033A/en active Pending
- 1993-05-19 EP EP93912658A patent/EP0641431B1/en not_active Expired - Lifetime
- 1993-05-19 ES ES93912658T patent/ES2109501T3/en not_active Expired - Lifetime
- 1993-05-19 WO PCT/DK1993/000171 patent/WO1993023738A1/en active IP Right Grant
- 1993-05-19 AT AT93912658T patent/ATE157771T1/en active
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6914250B2 (en) | 1997-03-07 | 2005-07-05 | Clare Chemical Research, Inc. | Fluorometric detection using visible light |
Also Published As
| Publication number | Publication date |
|---|---|
| ATE157771T1 (en) | 1997-09-15 |
| ES2109501T3 (en) | 1998-01-16 |
| DK66992D0 (en) | 1992-05-21 |
| NO944427D0 (en) | 1994-11-18 |
| EP0641431B1 (en) | 1997-09-03 |
| NO944427L (en) | 1994-11-18 |
| DE69313633T2 (en) | 1998-04-02 |
| EP0641431A1 (en) | 1995-03-08 |
| US5557415A (en) | 1996-09-17 |
| DE69313633D1 (en) | 1997-10-09 |
| JPH08504033A (en) | 1996-04-30 |
| DK0641431T3 (en) | 1998-03-30 |
| WO1993023738A1 (en) | 1993-11-25 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued |