Radiant energy – Luminophor irradiation – With ultraviolet source
Reexamination Certificate
1999-07-26
2002-05-21
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
With ultraviolet source
C250S461200, C250S370080, C385S120000
Reexamination Certificate
active
06392241
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns methods and apparatus for imaging, particularly the imaging of fluorescing samples of the type in which the sample is first illuminated with an excitation radiation such as ultra-violet light and is subsequently interrogated for any resulting emission light due to fluorescence from within the sample.
Typically a sample will comprise an array of a large number of sites each containing a small quantity of material or mixture of materials under investigation, and one convenient array comprises a plate containing a large number of wells arranged in a rectangular matrix each of which comprises a site; or a membrane, or a dish, over the surface of which exist a large number of reaction sites which are to be investigated.
For the purpose of investigation an image of each of the sites (preferably all at the same time) is formed at the input to a detector which converts light into electrical signals. The detector may comprise a CCD camera which may be cryogenically cooled or may comprise a CCD camera preceded by or including an image intensifying device.
BACKGROUND TO THE INVENTION
It is known from U.S. Pat. No. 4,922,092 to utilise a fibre optic device to couple the light emitted from such an array of sites to such imaging devices.
It is an object of the present invention to provide a device by which an array of such sites can be coupled to a source of excitation radiation (such as a source of UV light), as well as to the input of an imaging device, so that after exposure to excitation radiation, the sites can be inspected for any fluorescence arising therefrom.
The invention will be of particular benefit where the sites are small and closely packed together as in the case of high density multi-well plates.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a fibre optic coupling plate having a sample viewing face for receiving light from a sample, an output window for conveying sample originating (emitted) light to an imaging detector and an additional window through which excitation radiation can be projected, wherein there are provided a primary light path through the plate made up of optical fibres which will convey light entering the viewing face, directly and with minimal loss, to the output window, and a secondary light path separate from the primary light path, by which excitation radiation entering the additional window is conveyed to the viewing face for irradiating the sample.
The coupling plate preferably includes radiation reflecting or absorbing means to restrict the exit of excitation radiation therefrom except as required to irradiate the sample.
To this end, and where the material forming the coupling plate is not opaque to the excitation radiation, the optical fibres forming the primary path may be coated or sleeved with a material resistant to the transmission of the excitation radiation, and those areas of the output window not occupied by the optical fibre ends are rendered impervious to excitation radiation to prevent the latter from exiting in the direction of the detector.
The primary light path fibres are preferably formed from a material which readily transmits light emitted from the sample but only poorly transmits excitation radiation wavelengths.
The secondary path may comprise a second set of optical fibres which are bundled with the first set of fibres forming the primary path, and the fibres of both sets terminating in and comprising at least part of the sample viewing face.
The ends of the fibres from the two sets may be interspersed in a random manner across the face of the sample viewing window or may be arranged in a particular pattern so that the fibres of the first set bear a fixed relationship both in position and number relative to the fibres of the second set.
Where the second path is comprised of fibres, they are preferably formed from a material which readily transmits excitation radiation wavelengths.
In a preferred arrangement, the secondary path comprises selected optical material which is capable of transmitting the excitation radiation and which surrounds, but is optically screened, from the optical fibres which convey the image light to the output window.
The selected material is preferably in the form of a sleeve around each of the fibres of the first set, although in some circumstances it may be in the form of a block of material through which all the fibres in the first set extend.
In a particularly preferred arrangement, the coupling plate is formed from the said selected optical material and the fibres conveying the light from the sample to the detector are coated or sleeved with a material which is impervious at least to the excitation radiation wavelengths.
The optical screening is most simply provided by a coating the fibres making up the said first set. In known manner the coating material is selected so that it not only serves to reflect any internal radiation (so as to improve the efficiency of the fibres), but is also selected so as to be reflective at the wavelengths of the excitation radiation so that it not only prevents the latter from entering the fibres but also renders available and useful reflected excitation radiation for subsequent reflection and possible emission out of the coupling plate towards the sample as required.
By coating the fibres with a suitable material light within the fibres is completely prevented from escaping into the plate material surrounding them.
According to a particularly preferred aspect of the invention, each of the optical fibres in the first set is replaced by a bundle of parallel very small diameter fibres, each of which is coated as aforesaid to prevent the ingress of excitation radiation and the exit of light from within the fibre.
Preferably the small diameter fibres in each bundle are also separated one from the other by the said selected optical material which also surrounds and separates one bundle of fibres from adjacent bundles of fibres.
In one embodiment the plate includes a large number of separate fibres, (or fibre bundles) as aforesaid, distributed substantially uniformly across at least the input face area of the plate, each fibre or bundle of fibres extending from the input face to another face thereof, which serves as the output window.
If the density (ie the number per unit area) of the fibres (or bundles), across the face, is sufficiently high, it may not be essential for the fibres or bundles to be arranged in any particular pattern or have any particular spacing as between one and the next so long as each is a discrete isolated light path.
According to a further aspect of the invention, the input face area may be larger than the input window area so that the density of the fibres or bundles in the latter is greater than is the density of the fibres or bundles in the former.
According to a further aspect of the invention, where a coupling plate as aforesaid is to be used with a sample having a regular array of reaction sites, (or wells) then the fibres (or bundles of fibres) are preferably arranged (at least over the input face of the plate) in the same pattern and with the same interstitial spacing as are the sites in the sample, so that registration is possible between sites and fibres (or fibre bundles), one fibre (or fibre bundle) registering with each site.
Where the coupling plate is formed from selected optical material and the latter is used as the medium for conveying the excitation radiation, it is simply necessary for part of the well or site to communicate with a small region of the material forming the coupling plate to enable excitation radiation to exit thereinto.
Where each site is “seen” by one or a few large area fibres, the area of the end of each such fibre (or group of fibres) in the input face must be somewhat less than the area of the site or well with which it cooperates, so that at least part of each site registers with an area of the input face of the coupling plate not occupied by optical fibre(s) leading to the output window, so that excitation radiati
Hooper Claire Elizabeth
Neale Ann
Neale William Wray
Rushbrooke John Gordon
Gagliardi Albert
Hannaher Constantine
Lee Mann Smith McWilliams Sweeney & Ohlson
Neale Ann
Packard Instrument Company Inc.
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