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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/274—Calibration, base line adjustment, drift correction
  • G01N21/278—Constitution of standards
• • 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
• • 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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N2021/1748—Comparative step being essential in the method
• • 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
  • G01N2021/6417—Spectrofluorimetric devices
  • G01N2021/6421—Measuring at two or more wavelengths
• • 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
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
• • 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
  • G01N2021/6482—Sample cells, cuvettes
• • 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
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/274—Calibration, base line adjustment, drift correction
  • G01N21/276—Calibration, base line adjustment, drift correction with alternation of sample and standard in optical path

Definitions

• FLUORIMETERS This Invention relates to fluorlmeters.
• Fluorlmeters at present are laboratory Instruments of substantial size and use high power light sources such as 150 watt xenon lamp light sources and high voltage photomultiplier tube-detectors. Such Instruments while satisfactory are confined to use by skilled operators in laboratory conditions.
• fluorimeter which is not constrained to laboratory conditions.
• fluorimeter and analogous terms, used herein includes instruments using at least one of fluorescence, phosphorescence and luminescence and the like techniques including a calibration step using a fluorimetric standard, which are similar as will be understood by those skilled in the art.
• a method of flash fluorimetric analysis of a specimen expected to produce a first specific, fluorescent radiation on excitation including in the performance of a "blank” analysis: supporting at a test position a reference fluorophore expected to produce a second, characteristic, reference radiation on excitation, applying to said test position a pulse of excitation radiation, examining the radiation from the test position with a respective detector for the first radiation and a respective detector for the second radiation, determining from the relative response of the detectors a "blank” value for radiation from the test position in the absence of a specimen, and in the similar performance of a specimen analysis: supporting at said test position in the maintained presence of the reference fluorophore a specimen expected to produce said specific radiation on excitation by incident radiation, examining the radiation from the test position with said respective detectors, determining from the relative response of the detectors a specimen value for radiation from the test position in the presence of a specimen, and assessing said "blank" and specimen values to analyse the specimen with regard
• the reference fluorophore is incorporated in the construction of a cuvette to contain a specimen or in another form of specimen support.
• the cuvette may be of optical construction quality.
• the specimen for analysis may be a sample and a reagent to react to be capable of fluorescing to produce said specific radiation.
• One of the sample and the reagent may be present during the "blank” analysis the other being added after the "blank” analysis.
• the reference fluorophore may be included in one of the sample and the reagent.
• the method may include limiting the time between the reference and specimen analyses to ensure a stable reference fluorescent radiation.
• an instrument for fluorimetric analysis of a specimen is battery operated, and includes a control circuit and a flash tube and an optical path from the flash tube via a specimen position to photo detectors, the control circuit being effective to charge and fire the flash tube to provide appropriate radiation through the specimen position to provide in operation radiation including reference fluorescence to the detectors for processing by the control circuit to determine by ratiometric comparison, having regard to a calibration step, the level of specific radiation from a specimen at said specimen position.
• an optical structure formed in a body of material provided with apertures to receive and hold optical elements
• the body providing a specimen aperture to receive a specimen and hold the specimen in a specific position, a radiation source aperture to receive a radiation source and hold the source in a specific relation to the aperture, an input radiation path aperture to provide a specific path between the radiation source aperture and the specimen aperture and a detector aperture to define an output radiation path aperture to provide an output radiation path from the specimen aperture to radiation detectors.
• the body may be unitary, that is all 1n one piece.
• the body may provide a beam splitter aperture in the output radiation path aperture to receive and hold a beam splitter 1n a specific position 1n said output path and output radiation component apertures to define output radiation component paths from said beam splitter aperture, together with respective detector apertures in said radiation component paths, whereby the body of material around the apertures maintains a defined arrangement of optical elements with respect to said specimen aperture.
• the beam splitter may be of the partially-reflective type or of the fibre optic type.
• a body of material to contain an optical path arrangement, the body being of opaque material and defining apertures therein to receive and hold optical elements in said optical path arrangement, said apertures defined by said body including a specimen aperture to receive, in use, a specimen and hold a specimen in a specific position, a radiation source aperture to receive a radiation source and hold the source in a specific relation to the specimen aperture, an input radiation path aperture to provide a specific radiation path between the radiation source and the specimen aperture, an output radiation path aperture to define a specific output radiation path from the specimen aperture to a radiation detector aperture, whereby the body of material around the apertures maintains a defined arrangement of optical elements with respect to said specimen aperture.
• the apertures may have installed therein optical control elements to control at least one of intensity of radiation and frequency range of radiation.
• a fluorimeter optical core structure of unitary thermally conductive opaque material defining apertures for a radiation source, a specimen input radiation path, a specimen for examination and a specimen output radiation path, the body of material around the apertures maintaining said apertures, and optical elements therein, in a defined arrangement around the specimen aperture.
• Figure 1 shows a partial cross-section through the optical portion of a fluorimeter in accordance with the Invention, with the optical elements in diagrammatic form
• Figure 2 is a block diagram of electronic circuits associated with the fluorimeter of Figure 1
• Figures 3 and 4 show in partial cross-sections the optical portion of further fluorimeters in accordance with the invention.
• diagnostic chemical analysis potable water supply testing and environmental monitoring there 1s a growing need for On the spot' testing as well as the established centralised laboratory services.
• fluorimetry is a well-established laboratory technique but no instrumentlon Is available to enable On-s1te' or 'doctor's office' measurements to be made using fluorimetry.
• phosphorescence, delayed fluorescence and luminescence measurements are sufficiently similar to fluorescence to be included 1n the term fluorescence for the present description.
• a ratiometric dual wavelength fluorimeter and associated assay systems are now described.
• This fluorimeter measures fluorescence at two wavelengths from the same pulse of high intensity UV-VIS radiation.
• the construction of both portable battery operated fluorimeters and low cost laboratory fluorimeters 1s made possible.
• a device embodying the invention makes measurements by a ratiometric method using successive pulses of radiation passed through samples and thereafter measured at different wavelengths. Between these successive pulses the reaction of Interest 1s brought about and the successive measurements give information about the reaction.
• the method has calibration built Into 1t. This simplifies the device making field use possible. Laboratory use is not precluded, actually being of wider possibility, as such devices would be of lower cost and less demanding of operator skill than present devices.
• Figure 1 shows an outline, partly in cross-section, through a fluorimeter in accordance with the invention.
• a suitable framework (not shown in detail) is arranged to support the various elements.
• the details of construction will be readily apparent to those skilled in the art but significant points will be noted.
• the framework is a block of metal.
• a holder 51 for a cuvette 5 which is to contain a sample or other material, positions the cuvette precisely in an optical or other radiation path from a radiation source 1 to a detector assembly including specific radiation detector 10 and reference radiation detector 12.
• a source of reference fluorescent radiation is provided at the cuvette position, as described below.
• the radiation path includes apertures 2 and 3 and a quartz window 4.
• the source 1 is conveniently a high intensity xenon filled flash tube and if required a blocking filter 6 may be provided as shown between apertures 2 and 3, or elsewhere, to prevent decomposition of material in the cuvette by the flash energy.
• Filter 6 can be arranged to control the intensity and/or wavelength of the radiation reaching the cuvette.
• the cuvette 5 may be a precision quartz cell or may be of a disposable design, for example made by injection moulding from acrylic, polystyrene or other optical quality plastics material.
• the cuvette and holder should be marked to ensure re-insertion of the cuvette in the same relative position if removed.
• the path includes a quartz window 7 and a beamsplitter 8.
• Beamsplitter 8 may divide the radiation equally or be weighted (say 80/20) to increase sensitivity 1n one channel.
• Energy not deflected by the beamsplitter passes through a narrow band-pass filter 9 for the specific radiation and onto detector 10 for specific radiation.
• Energy deflected by the beamsplitter passes through a narrow band-pass filter 11 for the reference radiation and onto reference radiation detector 12 for reference radiation.
• the filters have appropriate wavelength or frequency response for the expected radiations. If the instrument is for a specific application a dlchroic filter could be used to maximise optical efficiency and eliminate the need for separate specific and reference radiation filters.
• the quartz windows retain liquid spilled from the cuvette within the cuvette holder.
• An optical link 13 such as a light pipe is provided between the reference detector 12 and the specific detector 10. This 1s to ensure that the specific detector always receives a constant part of the reference radiation.
• the reference radiation Is collected from the output side of filter 11 and applied to the detector 10.
• the structure of Figure 1 can be made 1n a very compact form.
• the outline 53 in one embodiment defines a 40 millimetre square plan view of a 40 millimetre aluminium cube in which the various elements are housed in cavities formed by milling and boring.
• the cuvette is basically the standard 10 millimetre square (Internal dimension) form, typically 12 millimetre square outside.
• the holder 51 can be a hole of diameter slightly less than the circumcircle of the cuvette plan with grooves reamed to allow the cuvette to enter only in the required position.
• the flash tube can be of the type used in pocket cameras and 1s some 35 millimetres long.
• the beamsplitter is readily obtainable from component suppliers such as Melles-Griot and the filters can be formed by grinding down available elements.
• the optical link can be a length of fibre optic.
• a main switch has two ganged elements 14A and 14B to switch the device ON and automatically set the ZERO for the device.
• Switch element 14A controls the action of a timer 15 which
• Switch element 14B when closed enables monostable 17 which is arranged to time a period sufficient for flash tube drive circuit 18 to
• ZES timing signals are derived from monostables 17, 20 and 30.
• Specific detector 10 and reference detector 12 are conveniently photo diodes and are connected to respective operational amplifiers 24 and 25.
• Gates 22 and 23 are respectively between the outputs of operational amplifiers 24 and
• An analogue divider 28 is arranged to receive the voltages on capacitors 26 and 27 and produce an output representing the ratio of these voltages.
• monostable 20 is connected to monostable 21 to provide a timing signal for a period during which gates 22 and 23 are open in response to the operation of monostable 17. This period occurs shortly after the flash tube is first fired, by monostable 17, and during it the voltage outputs of operational amplifiers 24 and 25 resulting from radiation incident on the photodiodes are sampled and stored on capacitors 26 and 27 respectively.
• the optical link 13 (Fig. 1) ensures that the specific detector always receives a constant part of the reference radiation.
• Monostable 30 is connected to operate gate 31 at the output of divider 28.
• the output of divider 28 is supplied both to gate 31 and to a first Input of a differential operational amplifier 36.
• the output of gate 31 is connected to a capacitor 32 at one input of a buffer operational amplifier 33.
• the output of operational amplifier 33 1s connected to a second Input of differential operational amplifier 36.
• the output of operational amplifier 36 is applied to a potential divider 40 and the tapped-off portion of the output displayed on indicator 37.
• the display on indicator 37 represents any difference between the inputs of differential amplifier 36.
• Switch 38 when closed enables monostable 39, which times the charging of the flash tube drive circuit 18 and then fires tube 1 in a manner similar to monostable 17.
• Monostables 20 and 21 respond to monostable 39 as described above for monostable 17 to sample and store the outputs on capacitors 26 and 27.
• Specific detector 10 and reference detector 12 will have responded to the radiation and the value of the ratio of the specific and reference radiation received is applied by the output of divider 28 only to the "first" input of differential amplifier 36. Any difference between the values of the inputs to differential amplifier 36 is shown on the display indicator.
• Timer 15 is effective to limit the time for the operation of switch 38. If switch 38 is not operated soon enough the cycle must be restarted with switch 14A/14B. Some five minutes 1s a suitable time limit for buffer amplifier 33 to hold the value acquired when the flash tube is first fired.
• cuvette 5 may be moulded as a disposable component from suitable plastics material.
• the optical requirements for the moulded cuvette for a fluorimeter according to the invention are not as severe as for disposable cuvettes currently in use. Only two clear faces are required and these will usually be adjacent. It is essential that if the cuvette is removed it 1s not turned round when It is replaced. Accordingly it should be marked or shaped to guard against this.
• the holder 51 may include a fillet in one corner to co-operate with a chamfered-off cuvette corner.
• a reference fluorophore can be incorporated in the cuvette itself, for example by including the fluorophore in the material from which the cuvette is moulded.
• fluorophore While the inclusion of the fluorophore in the cuvette is a very convenient arrangement other techniques may be used. For example a reference fluorophore may be included in a reagent put in the cuvette.
• band-pass filter 11 appropriate to the reference frequency from the fluorophone, for measurement by the detector 12 and simultaneously the other fraction to band-pass filter 9, appropriate to the specific frequency and which therefore blocks the reference frequency radiation from the fluorophore, for measurement by the detector 10.
• monostable 21 operates to open gates 22 and 23 for a sampling period of a few milliseconds to store the outputs of the operational amplifiers 24 and 25 on the respective capacitors 26, 27.
• band-pass filter 9 obstructs the reference radiation from reaching specific detector 10 but the optical link provides some radiation, as mentioned above.
• the delay can be chosen with regard to the type of measurement being made.
• the value representing the radiation ratio with a blank cuvette is thus held on capacitor 32 by operational amplifier 33 and applied to the second input of differential amplifier 36.
• the inputs to differential amplifier 36 are arranged to balance and a ZERO output is obtained on indicator 37 display. Suitable reset circuits may be needed for indicator 37.
• the "blank" in the cuvette is now replaced with the specimen for analysis so that the specimen analysis can be carried out.
• Switch 38 is operated and, provided timer 15 has not timed out, the flash tube fires as part of the continued automatic operation of the circuit.
• the excitation radiation passing into the cuvette now generates at least two fluorescent emissions when analyte is present, that is specimen generated fluorescence and reference generated fluorescence. Some of these emissions will pass through the quartz window 7 and thence into the beamsplitter 8. Fluorescence from the specimen is collected by detectors 10 and 12 and the ratio applied to the "first" input of differential amplifier 36. The optical link again provides some radiation to detector 10.
• the value of the ratio for the blank cuvette is already maintained at the second Input of differential amplifier 36 so the indicator 37 will display the difference between the value of the ratio for the specimen in the cuvette and the value of the ratio for the "blank" cuvette during the "blank” analysis.
• the scale length of indicator display 37 can be set by potential divider 40.
• Timer 15 ensures that the "zero" reading for the blank cuvette Is not invalidated by a delay before testing the specimen such that the "zero" Is no longer true because of instability in the instrument or the reference fluorophore.
• a fluorimetric standard such as a block doped with a fluorescent material 1n a known concentration to calibrate the device 1s well-known in the art and this step Is assumed to be carried out when needed, no specific mention being made.
• a blocking filter, 6, may be Incorporated to limit the excitation wavelength to wavelengths lower than the specific and reference emission wavelengths.
• the cuvette includes a fluorescent marker in the thermoplastic material from which it 1s moulded.
• a dual wavelength fluorimeter can be standardised (or normalised), sychronously with the test, as has been described above.
• the marker may be a phenol or indole , fluoresceine, quinine, anthracine, napthalone, ovalene, p-terphenyl , terphenyl-butodiene, rhodamine B, compound 610, or other similar compound.
• the marker may be available as a finely divided powder, it may be 'dusted' onto the natural granules, polystrene, polymethylmethacr late, TPX, or other optical grade thermoplastic.
• the marker may be dissolved in a solvent that is immiscible with the thermoplastic, the granules then washed in the doped solvent and air dried; this leaves each granule coated with a trace of fluorescent marker.
• the modified feedstock is then used to injection mould cuvettes.
• a simple mould may be fashioned, and cuvettes cast from acrylic or other polymer, where the monomer has been doped with small amounts of fluorescent marker.
• This technique 1s useful in optimising and testing markers.
• the cuvette 5 is a precision silica cell, for example to resist solvents
• the reference fluorophore can either be incorporated into one of the test reagents or doped into the silica or borosilicate material.
• Quinine sulphate is a suitable fluorophore for use in some reagents.
• the specific and reference wavelengths share a common optical path, and during the brief period of the excitation flash (1 to 5ms) the specific and reference detectors are responding synchronously to the same source radiation.
• the ratio:- Fluorescence specific /Fluorescence reference will be constant, regardless of variations in subsequent flashes of the excitation source.
• fluorimetry the channel in which radiation specific to the test is measured is synchronously normalised (or standardised) to the reference channel.
• the device described above is particularly suitable for battery-powered field use. It can be of compact, robust construction with a self-contained battery. The power consumption is relatively low. Power to charge the tube-firing circuits 1s needed for only a short time, for the rest of the time only the low power to operate the semiconductor control circuit and Indicator 37 is needed.
• the device 1s particularly suitable for use in the protocal devised by the American Society for Testing and Materials.
• FIG 3 this shows in Figure 3a a cross-sectional plan and in Figure 3b a cross-sectional elevation of another fluorimeter optical system mounted in a block of solid material by having the optical elements in bores through the block.
• the block indicated at 353, is conveniently a block 50 mm x 55 mm in plan and 35 mm thick of a readily worked material such as aluminium.
• the bores are cut into a solid block but other methods of construction, for example precision die-casting, may be used and the material also chosen for a particular purpose, so long as stability and accuracy are maintained.
• a holder is formed towards the middle of the block by a bore 351, of basically circular form with reamed grooves, in this case to receive a 16 millimetre square cuvette or other test piece or specimen carrier 305.
• a further bore 352 is made, parallel to the bore 351, to receive a radiation source 301 (shown diagramatically) which in this case is a xenon flash tube (specifically Maplin type FS77J).
• a radiation source 301 shown diagramatically
• This tube is designed to produce flashes at a fast rate typically 60 flashes per minute with an energy input of 0.3 watt seconds per flash.
• the tube is fitted into bore 352 by insulating bushes (not shown) and provided with suitable electrical connections.
• Bores 354, 355 and 356 are provided in a plane which Intersects with the bores 351, 352. These bores receive tubes which are fitted with various optical elements and the tubes then securely held in place in the required optical alignment, for example by grub-screws threaded into holes in the block, such as 358, 359. Conveniently the bores are 14 millimetres in diameter.
• the bore 354 this is provided with a filter and collimator assembly in the form of a tube, which is a good sliding fit in the bore, into which at each end is fitted an aperture disc (302, 303) while an ultraviolet pass filter 306 is held inside the tube at the end to be nearest the holder 351.
• the aperture disc 302, 303 may have circular apertures or slits or other forms of aperture to provide required collimation and intensity control of light from the radiation source 301.
• the ultraviolet filter 306 in this embodiment has a pass-band of 300 to 390 nanometres.
• This filter Is of a type used in diagnostic instruments such as that fitted to a TECHNICON (RTM) analyzer, and having a very well-defined pass-band peaking at about 356 nanometres, being reduced to about 10% transmission at 396 and 296 nanometres and having insignificant transmission at 428 and 260 nanometres.
• the assembly is positioned in bore 354 to be within 0.5 millimetres of a cuvette in holder 351.
• Bore 355 is at right angles to bore 354 and receives radiation emerging from holder region 351.
• a filter 371 to cut off ultraviolet radiation above 400 nanometres is fitted where bore 355 meets holder 351.
• this filter is a BALZERS (RTM) UV blocking filter.
• a quartz beam splitter 308, to extend into the UV range, is mounted in a tube 360 of 14 millimetre external diameter having a 10 millimetre hole at one end and a 10 millimetre hole In the side, positioned to match bore 356.
• the beam splitter is positioned in the tube with the reflection output face in the centre of and normal to the axis of the 10 millimetre side hole.
• Each detector assembly is a tube to fit the bore and having an Inner diameter to receive a respective photodiode 310, 312 supported by a sleeve.
• the photodlode is conveniently a Hammamatsu (RTM) type S1226-44BK which has a response range of 320 to 1000 nanometres.
• RTM Hammamatsu
• the respective interference filter 309, 311 (as described for Figure 1) is fitted in front of each diode, for example on the beam splitter cube faces as shown or on the diode Itself, and the detector assemblies are installed in the respective bores to just touch the beam splitter assembly. Electrical connections are provided for the photodlodes.
• Figure 4 shows in views 4a and 4b similar to 3a and 3b an optical system using optical fibres to split the radiation from the holder region, instead of a beam splitter.
• elements similar to those of Figure 3 are indicated by replacing the initial "3" with a "4" and the above description should be referred to.
• the beam splitter is installed in a bore 457 which is in the plane of bore 454 but at right angles to bore 454 to intersect the holder 451.
• a filter 471 has the same function as filter 371 (above).
• the beam splitter 1 s an ordered array of optical fibres all of which have one end exposed to the holder region.
• the fibres are distributed among two or more subsidiary bundles in a regular way and an individual photodetector and respective filter arranged for each subsidiary bundle of the other ends of the fibres.
• an ordered rectangular array 6 millimetres by 3 millimetres of 0.25 millimetre diameter fibres has alternate columns (6 millimetre dimension) allocated to each bundle (a 50/50 split).
• Suitable ferrules and epoxy based opaque potting compound are placed around the one end and other ends to hold the fibres in the required arrangement.
• the fibres at the one end and filter 471 are conveniently cemented into a tube 460 which can be fitted and held in bore 457 within 0.5 millimetres of a cuvette In the holder region.
• the ferrules at the other ends can be fitted into tubes which hold the respective interference filters and photodetectors (type as above) against the ends of the fibres.
• a bore is provided opposite bore 454 (or 354) to receive a detector by which radiation transmitted through the holder region can be examined.
• An alternative arrangement for the electronic control and measurement of the radiation source and resulting photodetector signals shown in Figure 2 is to use a microprocessor to control the firing of the flash tube and the processing of the detector signals. While the general procedure set out above still applies better control and performance is possible by the use of techniques to improve signal-to-no1se ratio.
• the microprocessor can quickly examine the "ratio" output. If this 1s not large enough to indicate an acceptable signal-to-noise ratio the flash tube can be fired again (within, for example, 300 milliseconds) and the signals summed on a statistical basis to improve the signal-to-noise ratio. This can continue at a flash rate of, say, 3Hz until an adequate signal has been acquired.
• the comparison of radiation intensities removes the effect of flash variation or other lack of optical reprodudblHty and "normalises' the response of the device.
• the measurement at two different wavelengths from a single pulse of light is to compensate for optical and electronic drift and noise in the measuring system.
• one of the wavelengths 1 s a test or active specific wavelength, which is that expected from the specimen to be analysed, whilst the other is a reference wavelength, produced by a fluorophore in the optical path, chosen such that response to this reference fluorescence is not affected by changes in the fluorescence intensity at the specific wavelength.
• Excitation with small flash tubes provides adequate intensity for silicon p.i.n. photodiodes, which are good detectors but need fairly high illumination levels.
• Disposable cuvettes, particularly if incorporating a fluorophore greatly ease the use of the device.
• To ensure that "drift" of electronic, optical or chemical characteristics is not significant once the device is normalised the specimen must be analysed within a set time.
• Dual wavelength fluorimetry will only give satisfactory performance when the detectors are working optimally. Too little illumination will give poor signal to noise and thus poor reproducibility. Excessive excitation energy will cause saturation and non-linearity.
• fibre optic beam-splitters may be extended further.
• a beamsplitter to divide the light from the flash tube to pass through different filters and then a similar fibre optic device to combine the light at the resulting selected frequencies for application to the cuvette and any contents can be excited simultaneously at distinct frequencies for detection in any convenient manner. In this way excitation in the UV range, below 300 nanometres, is possible.
• Suitable fibre optics and UV enhanced photodetectors can be employed.
• one bundle can convey light to a face of the cuvette holder and two other bundles, all three being combined at the cuvette face, convey light away from the same face to respective detectors, as set out above.
• this arrangement is useful for dry reagent chemistries where the reagent carrier can be inserted into a suitable form of cuvette holder or adaptor and measurement made on one face.
• Such chemistries are now being offered by, among other, Kodak Ektachem, Miles Laboratories (Ames Division), Boelwinger Mannheim, Fuji Film (all RTM).
• optical measurements are made through a transparent support layer either the support layer or the diffusely reflecting layer (T10 2 , BaS0 4 , etc.) may incorporate the reference fluorophore.
• the support is the reflective layer the measurement is made from above and either the diffusely reflecting support or the reagent zone may incorporate the reference fluorophore.
• Such techniques avoid the problems associated with the use of liquids in the fluorimeter and it is possible to use "dry” samples. Thus a form of "dip-stick” may be used.
• a clear plastics substrate has a fluorophore incorporated in it, possibly as described above for the cuvette, and a strip of substrate is coated with a reagent supported in a matrix, as is known 1n the art.
• the coated strip has the sample material applied to 1t in any convenient way, such as dipping or wiping, and 1s then placed in the holder in the fluorimeter.
• An adapter or spacing device may be used to ensure that the strip is 1n the correct position and does not touch the holder, to avoid contamination.
• the useful analytical range must conform to the optimum performance range of the photodetectors, and ideally, any critical analyte levels (e.g. clinical action levels, or statutary pollution limits) should occur at the peak performance level of the instrument. Methods having high intrinsic sensitivity could be accommodated by reducing the excitation pulse energy or by optically attenuating the radiation reaching the detectors.
• the two main types of fluorimetric assay are:

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