Imaging system for luminescence assays

Details Imaging system for luminescence assays Imaging system for luminescence assays

2001-01-03

2004-05-25

Bruce, David V. (Department: 2882)

Radiant energy

Photocells; circuits and apparatus

Optical or pre-photocell system

C250S227110, C356S319000

Reexamination Certificate

active

06740865

ABSTRACT:

FIELD OF INVENTION
This invention concerns methods and apparatus for imaging, and particularly the imaging of luminescence samples of the type in which the sample is illuminated with excitation radiation such as ultra-violet light, or where the sample is activated by some suitable chemiluminescent or bioluminescent means, and is interrogated for any resulting emission light due for example to fluorescence within the sample. The invention is particularly concerned with multipath (or multichannel) systems in which a large number of samples can emit light and need to be interrogated at the same time.
BACKGROUND TO THE INVENTION
PCT Application No. WO 98/0144 describes a fluorescence assay imaging system in which excitation radiation is supplied to the assay sample via an annular sleeve fitted around the end of a fibre optic bundle the end of which is in close proximity to the assay sample. The fibres collect emitted light due to fluorescence induced by the excitation radiation. It has been found that using an annular source of radiation does not produce the most uniform and sufficiently intense illumination of the assay reaction site, fan it is an object of the present invention to improve the uniformity and intensity of excitation illumination over the p resented area of each assay reaction site, without prejudicing any light collecting efficiency of the fibres.
Achieving uniformity over the reaction site has been found to be even more difficult to achieve where the sample is a very thin layer of cells or is contained or upon a thin gel or membrane.
The problems identified above become even greater as the area of each reaction site decreases. This is tending to occur as greater numbers of samples and therefore reaction sites, are accommodated in a sample supporting device such as a multi-well plate, multi-site membrane or gel or wafer, or chip of silicon or like material.
The invention endeavours to provide an improved illumination and collection system which allows sufficient excitation radiation to be introduced if required by the assay, and emitted light to be collected from reaction sites such as those in a 96 well plate for which the earlier imaging system of Wo 98/01744 is generally adequate, as well as the much smaller reaction sites such as now exist in high format multiple sample plates.
The invention is applicable to any luminescence producing assay.
Light emitting luminescence processes, including fluorescence, chemiluminescence and bioluminescence, and/or a combination of these processes, can be used in the measurements of biomedical and chemical assays. The wavelengths of excitation and emission for these processes are characteristic of the fluorescent and/or luminescent molecules and moieties being used. Wavelength ranges used are in the UV, visible, red and infra-red parts of the spectrum. A typical excitation range is 260-800 nm, a typical emission range is 320-1100 nm.
In the present application, the luminescent processes being measured include fluorescence, chemi- and bioluminescence.
In normal fluorescence, a fluorescent molecule or flurophore is excited by external radiation, such that it absorbs light energy and re-emits light at a longer wavelength.
The fluorescence may occur almost immediately, or later in time in which event it is referred to as time-delayed fluorescence.
In an alternative luminescent process, involving what is generally known as fluorescence or chemiluminescence energy transfer, energy is transferred from a donor molecule or moiety to an acceptor molecule or moiety, via a non-radiative mechanism. This mechanism can occur, eg via resonance or via electron transfer between atoms and molecules. Such luminescent donor and acceptor molecules may be fluorescent or chemi- or bioluminescent. The donor or acceptor molecules are generally different, and more than one molecule type may be used in either the donor or acceptor stage of the process.
The activation of the donor molecules may be via excitation light in the case of fluorescence or via chemical activation in the case of chemi- or bioluminescence. With fluorescence activation there may be a short delay between the excitation of the donor and the emission of the acceptor, in the range microseconds to milliseconds.
Energy transfer only takes place over very short distances (typically 10 nm and therefore the donor and acceptor molecules need to be in very close proximity. This can be achieved by direct bonding (eg covalent) of the donor and acceptor molecules, or linking of the two molecules by a biochemical bridge (eg via a peptide link). Alternatively, the molecules may be coated or bonded onto a solid phase, such that they are in close proximity (eg a microplate or bead). In a further example, the energy transfer from the donor may be via a reactive intermediate product, eg singlet oxygen or some excited chemical radical, which diffuses, eg in a fluid, to interact with the acceptor molecule.
Where no energy transfer takes place between the donor and the acceptor molecules, the donor molecule itself, when activated, will release energy directly as light, with emission wavelength characteristic of that molecule itself. When energy transfer occurs, the emission wavelength is characteristic of the acceptor molecule. Where the donor and acceptor molecules are different, the light emission from the acceptor molecules may be of a longer or shorter wavelength to the emission characteristic of the donor molecule.
When used in biomedical or chemical assays, to measure the presence or activity of a compound or agent, these luminescent processes may be used as an indicator of the presence or activity of such a compound or agent. The increase or decrease in light emission, from the donor or acceptor molecules, may be used as an indicator of the unknown compound being assayed. For example, the unknown compound might interact directly or indirectly with the energy transfer process, eg break the bridge between the donor and acceptor molecules or otherwise inhibit the transfer process. This would result in a change in the relative intensity of light emission of the donor and acceptor molecules, which could be detected by measurement, for example, at the two or more wavelengths which are characteristic of each molecule. Thus a ratiometric measurement involving various pairs of wavelengths characteristic of the molecules used may be appropriate.
PRIOR ART
EP 0580362 A1 describes a fluorescence detecting apparatus in which some of the fibres terminating below a sealed sample holder
7
convey excitation radiation to the sample, and others convey the fluorescence radiation away to a detector. On pages
3
and
4
, a preferred arrangement for weak fluorescence is described, in which the excitation fibres are concentrated in the centre of the bundle presented to the sample and those for receiving and conveying away from the fluorescence radiation are located annularly around the central excitation fibre core.
It was no doubt thought that by concentrating the excitation radiation fibres into a central region of the sample, and collating the weak emitted fluorescence from an annular region of the central core, much of the unwanted excitation radiation reflected or refracted back towards the fibres by the sample (or the reaction site), would thereby not be collected by the fibres leading to the detector.
However it has been found that this creates a virtual dead region in the centre of the reaction site where the product of excitation and light collection for any point is very low or zero (due to the annular arrangement of the collecting fibres), and genuine signals cannot be distinguished from background noise emanating from a large central area of the reaction site.
The present invention seeks to overcome this problem since for reliable and accurate assay evaluation, not only is it Li necessary for good uniformity of response to exist between one well and another over the entire well plate, but it is also very important that there is a high degree of uniformity of response across the area of each assay reaction

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