Patent Grant
6992769
1. Field of the Invention
The present invention relates to apparatus and to a method for carrying out optical analysis of samples and is applicable in particular to the analysis of biological, chemical and biochemical samples.
2. General Discussion and Related Art
Many chemical, biochemical and biological assays rely upon inducing a change in the optical properties of a biological sample being tested. For example, in order to test for the presence of an antibody in a blood sample, possibly indicating a viral infection, an enzyme-linked immunosorbent assay (ELISA) can be carried out which produces a visible coloured deposit if the antibody is present. An ELISA makes use of a surface which is coated with an antigen specific to the antibody to be tested for. Upon exposure of the surface to the blood sample, antibodies in the sample bind to the antigens. Subsequent staining of the surface with specific enzyme-conjugated antibodies and reaction of the enzyme with a substrate produces a precipitate which shows up the level of antigen binding and hence allows the presence of antibodies in the sample to be identified. This identification is usually carried out using a light microscope which allows an area of the substrate to be viewed by an operator.
In addition to colour staining using an ELISA, techniques such as fluorescence and gold labelling can be used to alter the optical properties of biological antigen material. Similar techniques are also used in general histology to visualise specific areas of tissue, e.g. particular cell types or cell structures, as well as in cell culture.
A significant disadvantage of existing optical analysis techniques is that they are open to human error because of their subjective nature. These techniques are also not suited to uses where a high throughput of samples is required, for example in blood screening applications or cervical smear tests, and are thus relatively expensive to use. The cost factor is exacerbated because, more often than not, different equipment is required for each particular technique.
An object of the present invention is to provide a technique for carrying out the optical analysis of samples which overcomes or at least mitigates certain of these disadvantages.
It is also an object of the present invention to provide an optical analysis technique which allows high speed automatic analysis of biological, biochemical and chemical samples and which is versatile enough to allow it to be used for a variety of different studies.
These objects are achieved by adapting the technology which has been developed in the field of audio and video compact discs to scan surfaces, to which a sample has been attached, using a light beam which is substantially focused onto that surface. A detector is arranged to detect light reflected from, or transmitted through that surface, and to determine from analysis of the detected light whether the light beam has been interfered with by the sample material.
According to a first aspect of the present invention there is provided a method of conducting an optical inspection of a biological, chemical, or biochemical sample, the method comprising the steps of;
supporting the sample on a substrate;
directing a beam of electromagnetic radiation from a radiation source onto the substrate;
scanning the beam over the substrate by rotating the substrate about an axis substantially perpendicular to the substrate and by moving the radiation source in a direction having a component radial to said axis; and
detecting radiation reflected from and/or transmitted through the substrate and sample and providing an output signal corresponding to the detected radiation.
According to a second aspect of the present invention there is provided a system for automatically carrying out an optical inspection of a sample to determine whether or not the sample comprises material which interferes with incident electromagnetic radiation, the system comprising:
a substrate having a surface for supporting the sample;
a source of electromagnetic radiation for providing a beam of electromagnetic radiation;
means for scanning said beam across said surface of the substrate; and
detector means which in use is arranged to detect electromagnetic radiation reflected from and/or passing through the substrate and the sample, the substrate being provided with distributed electromagnetic radiation modulating means for modulating at least a part of said beam with a digitally encoded position address indicative of the location on said surface on which the beam is currently directed, the detector means being arranged to decode the modulated electromagnetic radiation beam to determine the encoded address and to determine if the received beam has been modulated by any of said material which may be present in the sample.
The present invention enables the rapid scanning of a surface coated with components from a sample to determine their presence and also if necessary their optical properties. The system is particularly suited for carrying out the automatic inspection of samples with a high throughput. Moreover, provision of address information in or on the substrate enables the precise position of the electromagnetic radiation beam on the surface to be determined which in turn allows the accurate mapping of optical data, corresponding to attached material, to the surface. This enables regions of interest on the surface to be easily and quickly relocated.
The present invention is suited to carrying out ELISA where the specific antigen is coated onto the surface of the substrate. The surface is then exposed to the analyte and subsequently the specific enzyme and the resulting sample scanned to detect and quantify the enzyme linked to the surface. The system is also suited to carrying out histological analysis and to the quantitative study of gels run using electrophoresis.
Preferably, the electromagnetic radiation is light, e.g. infra-red, visible or ultra-violet.
According to a third aspect of the present invention there is provided a system for automatically carrying out an optical inspection of a sample to determine whether or not the sample comprises material which interferes with incident electromagnetic radiation, the system comprising:
a substantially planar substrate having a surface for supporting the sample;
a source of electromagnetic radiation for providing a beam of electromagnetic radiation;
means arranged on one side of the substrate for scanning the beam across the surface of the substrate;
a first detector for detecting electromagnetic radiation reflected from the substrate and the sample;
a second detector for detecting electromagnetic radiation passing through the substrate and the sample; and
control means coupled to the first and second detectors and for causing said beam to scan the surface of the substrate in dependence upon one or both of the outputs of the detectors and for detecting the presence of said components.
In a preferred embodiment of the above second aspect of the present invention the control means is arranged to determine the difference between output signals provided by said first and second detectors, which are representative of the signals detected, for the purpose of detecting said material without signal artifacts arising from, for example, dirt present on the side of the substrate opposite the support surface. The substrate may be provided with distributed address means for modulating the light beam with digitally encoded position information indicative of the area currently being scanned by the light beam, one or other of the detectors being arranged to decode the received light signal to determine the address of the location on which the light beam is incident.
According to a fourth aspect of the present invention there is provided a system for automatically carrying out an optical inspection of a sample to determine whether or not the sample comprises material which interferes with Incident electromagnetic radiation, the system comprising:
a disc comprising a plastic base layer on the upper surface of which is formed a plurality of perturbations, for interfering with incident electromagnetic radiation, representing digitally encoded data, and a surface for supporting the sample;
disc reading apparatus including a source of electromagnetic radiation for providing a beam of electromagnetic radiation, scanning means for scanning the beam across the upper surface of the disk, and an electromagnetic radiation detector for detecting radiation reflected from and/or transmitted through the disk and said sample components; and
means for rotating the disc about an axis substantially perpendicular to said beam,
wherein, in addition to being modulated by information digitally encoded onto the disc, the beam is additionally modulated by any of said material which is attached to the support surface of the disc.
Preferably, said electromagnetic radiation is visible light although infra-red or ultra-violet radiation may be suitable.
Preferably, the disc comprises a lower layer of transparent plastic on the surface of which is impressed, or otherwise produced, said digital information. This surface is coated with a partially reflective layer, for example of aluminium, which in turn may be covered by a further layer of transparent plastic.
In an embodiment of the above fourth aspect of the invention the upper surface of the disc is provided with a 3D surface topology arranged to provide growth and attachment cues for cells grown on the surface. For example, the surface may be provided with a rectangular grating for causing cells to align in a chosen direction. Alternatively, growth and attachment cues may be provided by chemical patterning of the surface, e.g. using fibrenectin, produced, for example, using photolithography.
In another embodiment of the above fourth aspect of the present invention the upper surface of the disc is coated with a gel suitable for carrying out electrophoresis on proteins, DNA etc. In order to run the gels radially, a first electrode may be provided at the centre of the disc with a second electrode being provided around the periphery of the disc. A well may be formed in the gel into which the analyte can be placed.
In order to calibrate a system embodying the present invention, the disc may be provided with a calibration track, e.g. a series of 256 grey levels. These levels may be printed onto the surface of the track using an ink jet printer.
According to a fifth aspect of the present invention there is provided a substrate for use in a system which is arranged to carry out an optical inspection on the substrate to determine whether or not material which interferes with incident electromagnetic radiation is present on a surface of the substrate, the substrate including a preformed calibration scale which enables calibration of said system.
Preferably, the calibration scale is a series of graded grey regions which reflect or transmit light to varying degrees. This scale may be printed on a surface of the substrate using an ink jet printer.
According to a sixth aspect of the present invention there is provided apparatus for conducting an optical inspection of a biological, chemical, or biochemical sample supported on a substrate, the apparatus comprising;
means for supporting a substrate and for rotating the substrate about an axis substantially perpendicular to the substrate;
a source of electromagnetic radiation for providing a beam of electromagnetic radiation;
drive means for moving the radiation source over the mounted sample in a direction having a component radial to said axis so that in combination with the means for rotating the substrate the radiation beam can be scanned over the substrate; and
detector means for detecting radiation reflected from or transmitted through the substrate and sample and for providing an output signal corresponding to the detected radiation.
For a better understanding the present invention and in order to show how the same may be carried into effect embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:
As discussed above, it is desirable to be able to optically scan a surface to which material is attached for the purpose of quantitative analysis of the material, or of a sample from which the material is derived or extracted.
If the system is used to carry out an ELISA, the appropriate antibody is bound to the upper surface of the disc (this may require some pre-preparation of the surface). The surface is then exposed to the sample to be tested, e.g. blood plasma, in order to bind any antigens in the sample to the antibodies. The surface is then washed to remove any excess, unbound, material and exposed to the appropriate enzyme-conjugated antibodies 5 which attach to the bound antibodies. The bound enzymes 5 can be visualised by reacting them with a substrate to produce a coloured precipitate. The precipitate presents a multiplicity of minute opaque patches over the upper surface of the disc. It will be apparent that the system is equally applicable to other types of assay which produce a colour, or other light interfering, change.
The disc is mounted on a rotatable shaft 6 which is driven by a drive means (not shown in
In operation, the disc is rotated by the drive means relative to the optical block. With the block stationary the light beam produced by the laser diode travels around a circular track of the disc. By moving the optical block along its linear track it is possible to scan the laser beam around any selected circular track of the disc. In areas of the disc surface where no light absorbing material is present, light passes through the upper plastic layer, is reflected from the metal layer, and travels back to the optical block, through the upper plastic layer. Light entering the optical block encounters first the quarter wavelength plat 37, which rotates the light by a further 45°, and then the polarising prism which, because the polarization of the reflected light has been rotated by 180°, causes the light to be redirected at right angles towards the detector.
When the light beam is incident upon areas of the disc surface where light absorbing material is present, light is absorbed by the material, both upon entry to and exit from the upper plastic layer of the disc, and the light received by the detector 11 is substantially reduced.
The detector 11 is coupled to a data analysis and logging system which stores the output of the detector. This system may store the output as a continuous analogue signal or as discrete digital samples which represents a reduced resolution image of the surface. Assuming the latter, the sampling rate may be varied to fit the data storage capacity available.
The optical, mechanical and electrical means so far described for implementing an analyte detector are substantially as conventionally used for reading data from compact discs. One such conventional system is described in the text book ‘Digital Audio and Compact Disc Technology’, 2nd edition, Edited by Luc Baert et al (Sony Service Centre Europe), Newnes, 1994.
The system of
Conventional compact discs encode digital information in an intermediate region of the disc by producing a series of perturbations 13 (e.g. bumps or pits) on the upper surface of a lower plastic layer 14 and subsequently coating this surface with a reflective layer 15 such as a thin layer of aluminium. The reflective layer is then covered with a layer of transparent plastic 16 which provides protection for the intermediate layer (FIG. 2).
It is possible to use this same technique to digitally encode position information into the disc of FIG. 1. Assuming that the position origin is at the centre of the disc, the first position on the innermost circular track or spiral can be imprinted with the position code zero (in binary representation). Position codes can be imprinted at discrete positions (e.g. every 2 to 3 μm or at any other appropriate interval) around that innermost track incrementing by one between each position. Similarly, the codes are incremented from track to track. Alternatively, address information may be distributed according to a track/sector arrangement in the same way in which servo-codes are encoded onto magnetic floppy and hard disks.
Over areas of the surface of the disc which are not covered by opaque material, light incident on the upper surface of the disc is transmitted through the upper transparent plastic protective layer and is incident upon the reflective layer. This light is reflected from the reflective metal coating except where that coating lies over a bump which causes incident light to be dispersed and not directly reflected back to the detector. The output from the detector can therefore be demodulated to determine the address of the disc surface which is currently being scanned.
Over areas of the disc surface where opaque material causes the incident light beam to be substantially absorbed rather than reflected, no position information will be present at the output of the detector. However, if the density of the opaque material is relatively low the gaps in the address information may not be significant.
In situations where address information is more critical however, a more sophisticated system can be utilised for which the optics are shown in FIG. 3 and which makes use of discs having address information digitally encoded and distributed over an intermediate layer as described above. This system also makes use of the fact that the reflective layer can be made to transmit a significant proportion of the incident light (e.g. 40%). As with the system of
The optical system of
Transmitted light which is not interfered with is received by a collimation lens 24, focused onto the upper surface of the disc, which directs the received light onto a partially transparent mirror 25 which in turn allows a fraction of the incident light to pass therethrough whilst causing the remainder to be reflected at right angles. Light passing directly through the partially transparent mirror is incident upon a further lens 26 which focuses the light onto the detection surface of a detector D2. Light reflected at right angles by the mirror 25 is incident upon a lens 27 which focuses light onto a detector D3.
As already described, a fraction of the light incident on the reflective layer within the disc is reflected back towards the first lens 22 which acts as a collimation lens directing light back to the polarising prism 21. The reflected light is now horizontally polarised and is reflected from the polarising prism at right angles to the optical axis. This reflected light is received by a fourth lens 28 which focuses received light onto a detector D1.
Light reflected by the reflective layer will be modulated with the information digitally encoded into the disc so that the output from the detector D1 will be similarly modulated. As this light does not exit from the upper surface of the disc it will not be interfered with by material attached to the upper sample support surface of the disc and address information can be determined from the output of D1 with minimal error.
Although not shown in
The output provided by detector D2 is modulated with the digital address information encoded onto the disc and, provided that no light absorbing material is attached to the upper surface of the disc, is substantially of the form of the output of detector D1, i.e. the ratio of the output signals of D1 and D2 will be constant. However, if light absorbing material is present on the upper surface of the disc this will interfere with light transmitted through the reflective layer and the output from detector D2 will drop whilst that from D1 will remain constant. The ratio of the output signals of D1 and D2 will change accordingly. If the material attached to the surface of the disc is reflective, e.g. gold labelled, the output of D1 will rise whilst that of D2 will fall when the light beam scans the material. The ratio of D1 to D2 will indicate the presence of such material.
In order to provide more stringent measure of the variations in the intensity of light transmitted through the disc the detector D3 is provided (although this is optional) which receives light from the partially transparent mirror through the aperture 53, lens 27 and pinhole arrangement 35. This arrangement effectively reduced the area of the disc surface from which light is received by the detector D3 and also reduces the depth of focus. If the output of detector D2, or the ratio D1:D2, exceeds a predetermined threshold the output of detector D3 can be used to increase the resolution with which the surface of the disc is viewed. The use of detectors D2 and D3 in combination prevents the likelihood of the detector D2 producing errors if the system used only detector D2. D3 may alternatively provide a second type of detector for detecting for example fluorescent light emitted by material attached to the surface of the disc.
The optical inspection system has a ‘U’ shaped arm 36 with a light source 52 and a detector 38 attached to the upper and lower ends of the arm respectively. The source and detector are connected to a laser controller 39 and a buffer 40, the latter being arranged to transfer detected signal data to a personal computer 41 via an analogue to digital converter 42 and a data store 43.
The disc 44 upon which the sample to be inspected is attached or supported is mounted on a rotatable spindle 45 which lies parallel to the bight 46 of the arm 36. The spindle 45 is driven be a spindle motor 47. The optical axes of the light source 37 and detector 38 are aligned with one another along the axis A—A.
The arm 36 is coupled to a stepper motor 48 which precisely rotates the arm in a plane parallel to the plane of the disc 44 such that, in combination, rotation of the arm and of the disc allows the light source/detector arrangement to be scanned across the entire useable surface of the disc. The stepper motor 48 is controlled by a motion controller 49, which in turn is controlled by the computer 41, such that the relative position of the spindle 45 can be determined to within an accuracy of 6 μm.
The disc is of a completely transparent material but is provided with a black bar 50 around a portion of its upper peripheral surface. The bar 50 acts as an angular calibration marking for the inspection system. When it is required to inspect a disc, the arm 36 is moved to an outermost “home” position, where the light source/detector arrangement is situated off the disc 44. In this position, the laser and detector are calibrated to ensure a constant, maximum, output signal. The arm 36 is then rotated to move the light source/detector arrangement towards the disc.
When the edge of the disc is detected, the arm is held stationary until the calibration marking 50 interrupts the beam. The leading edge of the marking 50 provides an origin to which the angular position of the detector can be referenced whilst the edge of the disc provides an origin for the radial position. Due to the accuracy of the stepper motor 48 and the spindle motor 46, it is then possible to precisely determine the position of the light source/detector arrangement relative to the disc.
In the system of
The system of
It will be apparent that various modifications may be made to the above described embodiments without departing from the scope of the invention. For example, the support surface of the disc may be scanned with infra-red or ultra-violet radiation rather than visible light. It is also possible to scan the surface with radiation which excites fluorescence in material attached to the surface and to use the detector (D2 or D3) arrangement to detect light at the emission wavelength.
It is also possible to construct the disc in such a way that the support surface (55) is internal to the disc (see
Another modification to the above described embodiments involves replacing the light detector with a photo-diode array, e.g. a CCD array. A preferred form of array is a linear array extending radially with respect to the disc. The light source would take the form of a laser line generator arranged to generate a radially extending line of light aligned with the diode array. Some degree of optical magnification may be incorporated between the source and the generator to allow the resolution of the system to be varied. After each rotation of the disc, the source/detector arrangement would be stepped inwardly by the length of the laser line. The advantages of this arrangement are higher speed and higher resolution.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9418981 | Sep 1994 | GB | national |
This application is a divisional of application Ser. No. 09/665,930, filed Sep. 20, 2000 now U.S. Pat. No. 6,327,031, which is a continuation of application Ser. No. 09/156,475, filed Sep. 18, 1998, now U.S. Pat. No. 6,256,088, which is a continuation of application Ser. No. 08/809,402, now U.S. Pat. No. 5,892,577, filed Jul. 28, 1997; which is a 371 continuation of PCT/GB95/02186, filed Sep. 15, 1995, which claims priority from UK Patent Application No. 9418981.8 filed Sep. 21, 1994.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3555284 | Anderson | Jan 1971 | A |
| 3736432 | Sweet | May 1973 | A |
| 3744975 | Mailen | Jul 1973 | A |
| 3798459 | Anderson et al. | Mar 1974 | A |
| 3966322 | Greaves et al. | Jun 1976 | A |
| 4363874 | Greenquist | Dec 1982 | A |
| 4478768 | Takeoka et al. | Oct 1984 | A |
| 4672600 | Balston et al. | Jun 1987 | A |
| 4687638 | Benajam | Aug 1987 | A |
| 4866688 | Ohtake et al. | Sep 1989 | A |
| 4876557 | Yabe | Oct 1989 | A |
| 5112134 | Chow et al. | May 1992 | A |
| 5122284 | Braynin et al. | Jun 1992 | A |
| 5132097 | Van Deusen et al. | Jul 1992 | A |
| 5173262 | Burtis et al. | Dec 1992 | A |
| 5199363 | Cheetham | Apr 1993 | A |
| 5265169 | Ohta et al. | Nov 1993 | A |
| 5270210 | Weyrauch et al. | Dec 1993 | A |
| 5281540 | Merkh et al. | Jan 1994 | A |
| 5310523 | Smethers et al. | May 1994 | A |
| 5329461 | Allen et al. | Jul 1994 | A |
| 5334837 | Ikeda et al. | Aug 1994 | A |
| 5407554 | Saurer | Apr 1995 | A |
| 5412087 | McGall et al. | May 1995 | A |
| 5413939 | Gustafson et al. | May 1995 | A |
| 5508200 | Tiffany et al. | Apr 1996 | A |
| 5510270 | Fodor et al. | Apr 1996 | A |
| 5585069 | Zanzucchi et al. | Dec 1996 | A |
| 5631844 | Margrey et al. | May 1997 | A |
| 5631166 | Jewell | Jun 1997 | A |
| 5684774 | Yamamuro | Nov 1997 | A |
| 5700655 | Croteau et al. | Dec 1997 | A |
| 5732066 | Moriya et al. | Mar 1998 | A |
| 5834758 | Trulson et al. | Nov 1998 | A |
| 5837552 | Cotton et al. | Nov 1998 | A |
| 5843655 | McGall | Dec 1998 | A |
| 5858804 | Zanzucchi et al. | Jan 1999 | A |
| 5863708 | Zanzucchi et al. | Jan 1999 | A |
| 5874219 | Rava et al. | Feb 1999 | A |
| 5876823 | Nagashima | Mar 1999 | A |
| 5882903 | Andrevski et al. | Mar 1999 | A |
| 5892577 | Gordon | Apr 1999 | A |
| 5909422 | Kamatani | Jun 1999 | A |
| 5922617 | Wang et al. | Jul 1999 | A |
| 5982535 | Inoue et al. | Nov 1999 | A |
| 5994150 | Challener et al. | Nov 1999 | A |
| 6030581 | Virtanen | Feb 2000 | A |
| 6117630 | Reber et al. | Sep 2000 | A |
| 6121048 | Zaffaroni et al. | Sep 2000 | A |
| 6192320 | Margrey et al. | Feb 2001 | B1 |
| 6225625 | Pirrung et al. | May 2001 | B1 |
| 6231812 | Rothberg et al. | May 2001 | B1 |
| 6327031 | Gordon | Dec 2001 | B1 |
| 6339473 | Gordon | Jan 2002 | B1 |
| 6342395 | Hammock et al. | Jan 2002 | B1 |
| 6387331 | Hunter | May 2002 | B1 |
| 6476907 | Gordon | Nov 2002 | B1 |
| 20020097658 | Worthington et al. | Jul 2002 | A1 |
| 20020098528 | Gordon et al. | Jul 2002 | A1 |
| 20020145960 | Worthington et al. | Oct 2002 | A1 |
| 20020163642 | Zoval et al. | Nov 2002 | A1 |
| 20020168663 | Phan et al. | Nov 2002 | A1 |
| 20020171838 | Pal et al. | Nov 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| 0 392 475 | Oct 1990 | EP |
| 0 417 305 | Mar 1991 | EP |
| 0 504 432 | Sep 1992 | EP |
| 0 521 421 | Jan 1993 | EP |
| 0866 449 | Mar 1997 | EP |
| 417305 | Oct 1934 | GB |
| 2 337 113 | Sep 1998 | GB |
| WO 9609548 | Mar 1996 | WO |
| WO 9635940 | Nov 1996 | WO |
| WO 9801858 | Jan 1998 | WO |
| WO 9807019 | Feb 1998 | WO |
| WO 9812559 | Mar 1998 | WO |
| WO 9815356 | Apr 1998 | WO |
| WO 9837238 | Aug 1998 | WO |
| WO 9913388 | Mar 1999 | WO |
| WO 9935499 | Jul 1999 | WO |
| WO 0005582 | Feb 2000 | WO |
| WO 0026677 | May 2000 | WO |
| WO 0206836 | Jan 2002 | WO |
| WO 0242498 | May 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20020135754 A1 | Sep 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09665930 | Sep 2000 | US |
| Child | 09991429 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09156475 | Sep 1998 | US |
| Child | 09665930 | US | |
| Parent | 08809402 | US | |
| Child | 09156475 | US |
