WO1998058252A1

WO1998058252A1 - An apparatus for measuring the fluorescence in a medium or a substance

Info

Publication number: WO1998058252A1
Application number: PCT/DK1998/000255
Authority: WO
Inventors: Hans Ole Nielsen; Jan Harries Hansen
Original assignee: Delta Lys & Optik
Priority date: 1997-06-16
Filing date: 1998-06-16
Publication date: 1998-12-23
Also published as: AU8011298A

Abstract

An apparatus with high sensitivity is provided for measuring the fluorescence in a medium. The apparatus comprises a pulsating light source (1), viz. a xenon flash lamp, emitting excitation light through a first filter system (3). The light is emitted from the first filter system into the medium through an optical light conductor (8) with a high numeric aperture, whereby the emitted fluorescent light returned from the medium is transmitted to a second filter system (10) through a second optical light conductor (9), also with a high numeric aperture. Both filter systems comprise optical filters. The emitted light is transmitted to a photomultiplier tube.

Description

1 Title: An apparatus for measuring the fluorescence in a medium or a substance.

Technical Field.

The invention relates to an apparatus for measuring the on-line fluorescence in a medium or a substance, the composition, structure and state of which are to be determined, and where a light source in the apparatus emits light through a first filter system at a wavelength interval determined by said filter into the medium or the substance, which returns an emitted light being filtered through a second filter system to a detector.

Background Art

The indicated apparatus for measuring the fluorescence is primarily intended for use within the biotechnology, articles of food, pollution control technology as well as the chemical industry. Other fields of application are, of course, also possible.

The conventional way of analysing for instance foodstuffs involves often a comprehensive destruction in form of a homogenization and extraction of the food followed by a chemical analysis. This destruction has inter alia the effect that data on chemical interactions and the structure and state of the food are lost.

During recent years, spectral analysing techniques and picture analysing methods have been developed which allow a measuring directly on the food as it is and consequently also a good determination of the composition, structure and state of said food. Subsequently, it is possible to process these data by means of modern computer technology.

During the 19 'eighties, near infrared spectroscopy was used for instance for determining the content of starch and proteins in for instance grain samples. This near infrared technique allowed a detection of signals from chemical components, such as the above starch and proteins, which are often present in high concentrations in the food. If fluorescence spectroscopy is used instead it is possible to detect signals from low concentrations of substances, such as for instance from flavonoides, amino acids, phenolic components and NADH. The fluorescence technique is up to a 1000 times more sensitive than the near infrared technique. The fluorescence technique provides furthermore a so-called spectral "finger-print" of the food, but the fluorescent "finger-print" is based on data from another type of chemical components compared to the near infrared "finger-print" . Thus the analogy to the near infrared technology is obvious, and accordingly the fluorescence spectroscopy can be used in the same way as the near infrared technology for predicting other types of quality parameters.

An example of an apparatus employing this known fluorescence spectroscopy technique is indicated in the preamble of claim 1. However, it should be fully realized that in such an apparatus the medium or substance, the composition, structure and state of which are to be determined, is subjected to a light which in addition to the returned emitted light also involves returned reflected light which may be 10⁶ times stronger than the emitted light to be measured on by the apparatus, said returned emitted light being fluorescent light at a slightly increased wavelength.

Brief Description of the Invention

Therefore the object of the invention is to structure the apparatus indicated in the introduction in such a manner that it possesses a maximum sensitivity towards the emitted light without the directly reflected light interfering with the measuring.

The apparatus according to the invention is characterised by comprising as light source a pulsating light source, viz. a xenon flash lamp, by both the first filter system and the second filter system comprising optical filters, and by the light being emitted from said first filter system into the medium or the substance through an optical light conductor with a high numeric aperture, whereby the emitted fluorescent light returned from the medium or the substance is transmitted to the second filter system 3 through a second optical light conductor also with a high numeric aperture.

The combination of members indicated in the characterising clause of claim 1 is thus essential to the obtained technical effect. Thus a pulsating light source, viz. a xenon flash lamp, is used which provides a very strong light in the entire ultra-violet and visible wave range. In addition, this type of lamp has a very long life and involves a minimum heating. The previously known technique usually employed a continuous mercury lamp which limits the use to nothing but the well-defined spectral lines. Furthermore, this light source involves much heating and accordingly it has a relatively short life. As both the first filter system and the second filter system employ optical filters it is possible to pass much more light, i.e. up to 10 to 30 times more light through said filter systems than by means of an optical grating. The resulting apparatus is very sensitive to the weak fluorescent signals. The preferred spectral range of the excitation radiation is from 250 nm to 580 nm. Correspondingly, the preferred range of the emission radiation is from 270 run to 600 nm. The band width of the excitation filters and the emission filters, respectively, is in the preferred embodiment 20 nm. Previously, the ultra-violet radiation in these ranges necessitated the use of quartz fibres either one by one or in bundles. Previously it was furthermore necessary to use quartz fibres which have a very small opening angle of approximately 25 ° , and as the collectable energy is dependent on the opening angle square it is very important that the apparatus according to the invention comprises optical light conductors with a high numeric aperture, viz. a very large opening angle. The presently preferred light conductor is a liquid-filled light conductor of a diameter of 3 mm. This type of light conductor has an opening angle of 50 to 70° in comparison with the 25° of a quartz fibre. As the collecting efficiency is propor- tional to sinus to half the angle square this means that this type of light conductor with a substantially enlarged numeric aperture increases the efficiency by 4 to 7 times of both the excitation channel and the emission channel. As the structure of this type of fibres does not involve a packing loss, viz. an interspace between the fibres which are glued together into a bundle, the transmission through the liquid-filled fibres is typically 50% higher than through ordinary quartz fibres within the ultra-violet and visible spectral range. All things considered the above ensures an improvement of the sensitivity by approximately 12 to 21 times compared to a bundle of quartz light conductors. Another advantage obtained by using the described light conductor rather than a bundle of quartz light conductors is that the emission of fluorescence from the fibre itself, i.e. including the glue and possible impurities, is significantly reduced. In comparison with the bundles of quartz fibres, the liquid-filled light conductors are less expensive in the same dimensions.

The previous use of gratings excluded the utilization of the large numeric aperture in the light conductors. In addition, problems are attached to obtaining a sufficiently efficient filter system when said filter system is based on gratings because in some situations the detector is subject to emissions not deriving from the fluorescence, but from reflected light. Therefore the invention ensures an optimum situation by using filters in combination with light conductors with a high numeric aperture. In addition, the filters possess very good blocking properties with the result that they only allow passage of fluorescent light and efficiently block die reflected radiation unlike gratings. The gratings allow also passage of ordinary light with the result that the entire measuring is blurred because nothing but reflected light is detected unless two gratings are used in sequence, viz. a double monochromator. Therefore the particular combination of a filter with a high numeric aperture and optical light conductors as well the pulsating light source is instrumental in obtaining a sensitivity not being achieved by means of any other structure. The resulting apparatus is therefore particularly suited for measuring strong turbid media, i.e. absorbing media containing a high concentration of solid substance and many particles.

It should be noted that the apparatus also allows an omission of the first filter system or the use of a completely neutral filter with the result that nothing but a white light is emitted into the medium or the substance. As a result, it is possible to measure the reflectance and transmittance of the medium or the substance. Such a measuring provides also very useful data compared to the fluorescence measuring. Measurings of the reflectance and the transmittance provide further data on the composition of the substance in addition to the amount of data provided by the fluorescence measuring alone.

Brief Description of the Drawings

The invention is explained in greater detail below with reference to the accompanying drawing, the sole Figure of which shows a drawing in principle of the apparatus according to the invention for measuring the fluorescence.

Best Mode for Carrying Out the Invention

The Figure shows a light source in form of a xenon flash lamp 1 , which is fed with power from a high-voltage power supply 2. The light source 1 emits a pulsating white light hitting a first filter system 3. The first filter system 3 comprises a filter wheel 3 turned stepwise about a shaft 6 by means of a step motor 5. The filter wheel 4 comprises a number of single filters 7 equally spaced from the shaft 6 in such a manner that each single filter 7 can be hit by the white light from the light source 1 when the filter wheel 4 is caused to stop by means of the motor 5. An optical light conductor 8 is accommodated on the side of the light conductor 1 which faces the filter wheel 6. This optical light conductor 8 is hit by the filtered light from the filter 7, said light being within a predetermined wavelength range. This light, viz the excitation light, is conducted through the light conductor so as subsequently to hit the medium or the substance, the composition, structure and state of which are to be determined, the latter not appearing from the drawing.

The light is returned from the medium or the substance both in form of reflected light and in form of emitted light, i.e. fluorescent light at a slightly higher wavelength compared to the reflected light. The latter emitted light is, however, far weaker, often perhaps 10⁶ times weaker than the reflected light. The returned light is received by a second optical light conductor 9. Both the first optical light conductor 8 and the second optical light conductor 9 have a high numeric aperture, and the presently preferred light conductors are of a type filled with liquid, viz. liquid light guides. Such light conductors present an opening angle of approximately 50 to 70°. The returned light, viz. both the reflected light and the emitted light, is emitted towards a second filter system 10. This second filter system 10 comprises a second filter wheel 11 , which can be turned stepwise about a shaft 13 by means of a second motor 12 in such a manner that the light from the optical light conductor 9 in each of these steps hits an optical filter 14. This optical filter is arranged in the filter wheel 11 in the same manner as the filters 7 in the filter wheel 4. The filter system analysing the light from the emission channel must be able to receive the light at the same angular scattering. A filter is therefore used. An outstanding advantage is found in using optical filters for structuring an optical system because such an optical system can accept the large angles presented by the above optical light conductors with a high numeric aperture. In addition, these optical filters possess very good blocking properties with the result that they can allow passage of the fluorescent light and efficiently block the reflected radiation. If gratings had been used it would have been impossible to utilize the large numeric aperture of the light conductors, and furthermore problems would have arisen in making the filter sufficiently efficient. Subsequently, the light from the respective optical filter 14 hits a photomultiplier tube 15. This light has been subjected to an efficient filtration with the result that the reflected light hits the photomultiplier tube and interferes with the measuring.

The Figure shows furthermore a flow chart of a number of units serving to control the motors 5, 12, the power supply 2 to the light source 1 and the photomultiplier tube 15. These members comprise a filter control means 16, a connecting module 17, a detector amplifying module 18, a detector control means 19 and a sensor control means 20. Moreover a power connection 21 is provided which feeds power to the individual units. Finally these units co-operate with a data collecting and controlling unit not shown in the Figure, but the connection of which is indicated by means of the reference numeral 22.

A measuring by means of the apparatus according to the invention renders it possible 7 to obtain a number of measuring locations. The latter is obtained by the data collecting and controlling unit or the controlling computer initially allowing the first filter system to be idle while one filter 7 thereof is operating at the same time as the second filter system 10 is turned stepwise while each filter 14 thereof is able to emit light in sequence to the photomultiplier tube 15. Then the first filter system is moved to a new stationary position, whereafter the second filter system 10 again can receive a number of measuring locations. This procedure is followed for all the filters 7 of the first filter system 3.

It is also possible to measure the reflectance or the transmittance by means of the apparatus. If the first filter system 3 is omitted or if the operating filter of the filter wheel 4 is replaced by a neutral filter, it is possible to measure the reflectance and the transmittance of the reflected light. In this manner other data on the composition of the substance are obtained.

Claims

8Claims

╬æn apparatus for measuring the fluorescence in a medium or a substance, the composition, structure and state of which are to be determined, and where a light source in the apparatus emits light through a first filter system at a wavelength interval deter- mined by said filter into the medium or the substance, which returns an emitted light being filtered through a second filter system to a detector, characterised in that the apparatus as light source comprises a pulsating light source, viz. a xenon flash lamp, that both the first filter system and the second filter system comprise optical filters, and that the light is emitted from the first filter system into the medium or the substance through an optical light conductor with a high numeric aperture, whereby the emitted fluorescent light returned from the medium or the substance is transmitted to the second filter system through a second optical light conductor also with a high numeric aperture.

2. An apparatus according to claim 1, characterised in that the individual filters of each of the two filter systems are mounted in respective filter wheels, which are coupled in the respective light paths by means of the motors of the first and the second filter system, respectively, and in accordance with a predetermined program.

3. An apparatus according to claim 2, characterised in that it comprises a computer, which in accordance with the predetermined program both turns the filter wheels with the optical filters into the respective light paths by means of the motors and register the measuring results so as to provide a measuring characteristic which is an exponent of the composition of the medium or the substance.

4. An apparatus according to any of the preceding claims, characterised in that the detector receiving the returned emitted light filtered through the second filter system is a photomultiplier tube.

5. An apparatus according to any of the preceding claims, characterised in that the aperture of the light conductors exceeds 0.2.