FLUORIMETRIC ARRANGEMENT

Abstract: A fluorimetric arrangement includes a sample support (15), a source of incident light (12), a detector (16) of fluorescent light emitted by the sample, and a filter (22), for separating the fluorescent light from the incident light reflected from the sample. There may also be a support (14) for a test sample and an optical component (20) adjustable so that the incident light is directed selectively onto the reference sample (10) or the test sample (11), and the respective fluorescent light directed to the detector (16).

Description: "FLUORIMETRIC ARRANGEMENT" This invention relates to a fluorimetric arrangement which has particular though by no means exclusive application to fluoroimmunoassay measurements. The practice of utilizing fluorophore-labelled antibodies as tracers is well established in immunological assay systems, but identification of the residual fluorophore-labe led antibodies or proteins is effected by fluorescence microscopy and therefore relies critically on the colour and spatial eye discrimination ability of the user. The recent introduction of time-resolved fluoroimmunoassay for discriminating otA: background fluorescence originating from non-fluorophore-labelled proteins has permitted the development of quantitative procedures but fluorimeters marketed for this purpose have been suitable for handling only liquid media and are not adaptable to the examination of multiple samples on paper or plastic substrates. The favoured approach for high speed fluoroimmunoassay is to bind the residual fluorophore-labelled entity to such a substrate, which may typically be a membrane structure. A particular difficulty in making quantitative measurements with samples on substrates, and on paper substrates in particular, is the high proportion of reflected incident light which must be discriminated from the fluorescent light. Paper tends to diffusely reflect the light with the same angular distribution expected from the fluorescence. It is an object of the invention to at least partly meet this difficulty. The invention provides in one aspect a fluor- imetric arrangement comprising: a sample support; a source of incident light; a detector of fluorescent light emitted by the sample; and means to position filter means for separating the fluorescent light from the incident light reflected from the sample. In a typical case, the fluorescent light will be of a higher wavelength than the incident light and the filter means may then comprise dichroic filter means. The filter means may be a bandpass interference filter selected to transmit incident and reflected incident light and to reflect the fluorescent light at a finite angle to the incident light. Preferably, the bandpass interference filter is arranged with respect to the reflected incident light so that the latter has the smallest angle of incident thereto consistent with adequate and practical angular separation of the fluorescent light. Such angle of incidence may conveniently be in the range 10 to 20°. Said filter means may comprise part of said fluorimetric arrangement. In a second aspect, the invention further provides a fluorimetric arrangement comprising: respective means to support a reference sample and a test sample; a source of incident light; a detector of fluorescent light emitted by the samples; and optical means adjustable so that the incident light is directed selectively " onto the reference sample or the test sample, and the respective fluorescent light directed to the detector. The aforesaid adjustable optical means may conveniently comprise a pivotable mirror which may be arranged for oscillation to alternately and periodically direct the incident light onto the reference sample and the test sample and receive the respective fluorescent light for direction to the detector. Such a mirror is preferably arranged in the optical path between the two samples and filter means for separating the fluorescent light from incident light reflected by the respective sample. It will be understood that the invention in either of its aspects may include appropriate optical devices for defining the respective light paths. Such devices would typically include one or more lenses, and a filter associated with the detector. It will be further understood that a phase sensitive arrangement may be employed for enhancing the detected signal: this may be provided, for example, by pulsing or mechanical chopping of the incident light or, in the second aspect of the invention, by oscillation of the mirror through a condition in which there is no light path between either sample and the detector. The invention will be further described, by way of example only, with reference to the accompanying schematic drawing of a fluorimetric arrangement embodying both aspects of the invention. The illustrated arrangement includes a lamp source 12 of incident light 5, respective sample supports 14, 15 for a reference sample 10 and a test sample 11, a photovoltaic detector 16, and a set of optical devices indicated generally at 18 for defining alternate incident light paths from lamp 12 to the samples and fluorescent light paths from the samples to detector 16. Optics 18 includes a rotatably oscillatable mirror 20 which performs the dual function of directing incident light 5 selectively onto reference sample 10 or test sample 11 and chopping the incident and fluorescent light for phase sensitive detection. Further components of optics 18 are a dichroic filter 22 which acts as a beamsplitter to separate the fluorescent light 6 from the reflected incident light, a trio of lenses 24, 25, 26 arranged to collimate various light beams at filter 22, and a detection enhancement or analysis filter 28 in front of detector 16. Collimated light is preferred at filter 22 in the case where sample 10 includes a paper substrate in order to reduce spectral overlap of the input and output beams. For a transparent substrate, e.g. a plastic substrate, some overlap of the input and output beam is tolerable and collimation is therefore not as critical. The three lenses may then be substituted by a single lens, for example in the region of lens 25 in the drawing. Dichroic filter 22 comprises a bandpass interference filter selected to transmit the incident and reflected incident light but to substantially wholly reflect the fluorescent light 6 to detector 16. It is interchangab from measurement to measurement and is held in a mount indicated at 23. Filter 22 is positioned so that the angle of incidence θ^ of the reflected incident light is at a minimum consistent with adequate separation of the fluorescent light and practical physical arrangement of the parts. A suitable angle is 10° and it is preferably not greater than 20°. Instead of using the oscillating mirror for phase-sensitive detection, a simple motor driven mechanical chopper may be positioned between lamp 12 and filter 22. Lenses 24, 25, 26 may be large plastics types as the formation of a good image at the detector is not critical. Collection over a f/1.5 cone would be highly satisfactory. The image size formed on the paper sample may typically be of the order of 10mm by 2mm, with an illumination angle of about 15°. EXAMPLE The application of the illustrated fluorimetric arrangement to a typical fluoroimmunoassay system will now be described. A widely used fluorophore tracer in immunoassays is fluorescein isothiocyanate (FITC), commonly referred to as fluorescein. This tracer has an absorption band peaking just under 500nm and a fluorescence band with a peak just under 530nm. The detection enhancement or analysis filter 28 was selected as Schott glass type OG530, which transmits light above about 520nm. The bandpass interference filter chosen as dichroic 6 filter 22 was IFV2 which exhibits transmittance over a narrow band between about 430 and 470nm and was thus reasonably effective in reducing reflected light in the detected signal. An ideal bandpass interference filter for this application would be centred in the range 460 to 480nm with a negligible transmittance at wavelengths longer than 500nm. A "square" bandpass filter rather than a conventional filter wave stack or an induced transmission type should have the required rejection. Two nominally identical filters used in parallel could be used to improve the rejection ratio. Some 8% of the reflective input light would be reflected to the detector if the outer surface of the filter were uncoated. The source was a quartz-halogen lamp running at a measured colour temperature 2880°K. The detector was a UDT Pin - 10D silicon photodiode, used in photovoltaic mode. The described arrangement has been advanced merely by way of explanation and many modifications may be.made thereto without departing from the spirit and scope of the invention which includes every novel feature and combination of novel features herein disclosed.

Claims: CLAIMS: 1. A fluorimetric arrangement comprising: a sample support; a source of incident light; a detector of fluorescent light emitted by the sample; and means to position filter means for separating the fluorescent light from the incident light reflected from the sample. 2. A fluorimetric arrangement comprising: a sample support; a source of incident light; a detector of fluorescent light emitted by the sample; and filter means for separating the fluorescent light from the incident light reflected from the sample. 3. A fluorimetric arrangement according to claim 1 or 2 wherein said filter means comprises dichroic filter means. 4. A fluorimetric arrangement according to claim 3 wherein said filter means comprises a bandpass interference filter selected to transmit reflected incident light and to reflect the fluorescent light at a finite angle to the incident light. 5. A fluorimetric arrangement according to claim 4 wherein said bandpass interference filter is arranged with respect to the reflected incident light so that the latter has the smallest angle of incidence thereto consistent with adequate and practical angular separation of the fluorescent light. 6. A fluorimetric arrangement according to claim 5 wherein said angle of incidence is in the range 10 to 20°. 7. A fluorimetric arrangement according to any preceding claim wherein said filter means is disposed in the optical path between the light source and the sample support. 8. A fluorimetric arrangement according to any preceding claim further comprising a second sample support, said sample supports being for a test sample and for a reference sample, and optical means adjustable so that the incident light is directed selectively onto the reference sample or the test sample, and the respective fluorescent light directed to the detector. 9. A fluorimetric arrangement according to claim 8 wherein said optical means comprises a pivotable mirror arranged for oscillation to alternately and periodically direct the incident light onto the reference sample and the test sample and receive the respective fluorescent light for direction to the detector. 10. A fluorimetric arrangement according to claim 9 wherein said mirror is arranged in the optical path *» between the two samples and said filter means. 11. A fluorimetric arrangement according to claim 9 or 10 wherein said mirror also serves as a phase sensitive arrangement for enhancing the detected signal, by being oscillatable through a condition in which there is no light path between either sample and the detector. 12. A fluorimetric arrangement comprising: respective means to support a reference sample and a test sample; a source of incident light; a detector of fluorescent light emitted by the samples; and optical means adjustable so that the incident light is directed selectively onto the reference sample or the test sample, and the respective fluorescent light directed to the detector. 13. A fluorimetric arrangement according to claim 12 wherein said optical means comprises a pivotable mirror arranged for oscillation to alternately and periodically direct the incident light onto the reference sample and the test sample and receive the respective fluorescent light for direction to the detector. 14. A fluorimetric arrangement according to claim 12 or 13 wherein said mirror also serves as a phase sensitive arrangement for enhancing the detected signal, by being oscillatable through a condition in which there is no light path between either sample and the detector.

Inventors: GARDNER JAMES LAURIE, GARDNER, JAMES, LAURIE
Applicants: COMMW SCIENT IND RES ORG, COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH OR
Application year: 1986
Main classification: G01N21
IPC codes: G01N21