Imaging system for fluorescence assays

Details Imaging system for fluorescence assays Imaging system for fluorescence assays

2000-02-15

2003-04-29

Evans, F. L. (Department: 2877)

Optics: measuring and testing

By shade or color

With color transmitting filter

C250S458100

Reexamination Certificate

active

06556299

ABSTRACT:

FIELD OF 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 within the sample.
BACKGROUND TO THE INVENTION
U.S. Pat. No. 4,922,092 describes a fibre optic device used to couple the light emitted from an array of well sites in a plate to an imaging device.
Our concurrent International Patent Application No. PCT/GB97/01825, filed Jul. 4, 1997, describes alternative arranges and systems incorporating fibre optic devices by which a large number of well sites can be inspected for fluorescence arising therefrom.
It is an object of the present invention to provide an improved optical fibre transfer device and an improved method of coupling the output of such a device to a camera input.
In FIGS. 10 and 11 of our concurrent Application, the output end of the fibre optic plate is shown coupled to a camera input window via a filter. The purpose of the filter is to restrict wavelengths entering the camera to those of expected, or wanted, emissions so as to ideally remove from the camera input any radiation at unwanted wavelengths such as stray excitation radiation transmitted via the fibre optic plate, or the like.
In an example involving 96 fibre optic bundles, the 96 outputs of the bundles may be arranged in any convenient configuration or aspect ratio depending on the output of the camera to which the image is to be applied. Where the latter is generally circular, the 96 rods may be arranged in a generally circular or hexagonal array so as to substantially fill the entrance window of the camera, and if the latter is 40 mm diameter and the rods fill an area of 32×42 mm
2
1
, there should be adequate spacing between rod centres (approximately 2.5 mm) to ensure minimal cross-talk between bundles. Where an emission filter is inserted between the plate and the camera input, this should be as thin as possible, and may need to be less than 0.5 mm to ensure acceptable levels of cross-talk.
Since the excitation wavelengths may be very close to the wavelengths of emitted radiation from the samples or where two or more emitted radiations can arise and selection as between one wavelength and another is desired, the difference between the wavelengths of the different emitted radiations may be very small. In these circumstances ordinary filters may not be sufficiently selective.
THE INVENTION
According to the present invention in an imaging system for fluorescence assays, a fibre optic coupling plate for transmitting radiation emitted by a sample towards a camera, is combined with an interference filter so as to enable highly selective transmission of radiation to the camera, according to wavelength.
The present invention also envisages the combination of an interference filter with a modified fibre optic coupling plate such as described herein, in which the sample sites or wells of an array thereof are viewed by separate fibre optic bundles, and each bundle transmits emitted light from a one sample or well to a discrete region of the field of view of the camera.
Typically the interference filter is located between the output end of the fibre optic coupling plate and the camera input window.
Where a system is intended to operate at a single wavelength, the interference filter may form part or comprise the input window of the camera.
Where, as is more likely, the system is to be capable of handling different assay chemistries and selection of different wavelengths, provision may be made for interchanging the filter so as to render the camera more or less sensitive to different wavelengths.
In one arrangement, two or more interference filters may be located in apertures in a slidable or rotatable support plate located between the output side of the fibre optic coupling plate and the input window of the camera, and the filter support plate filters is movable so as present one or another of the filters to the camera as appropriate.
The movement may be effected manually, or drive means may be provided, to effect the movement to position different filters in place.
Where the inspection system is computer controlled, the control system may be programmed to move the filter support plate according to the assay chemistry and/or wavelengths involved, or may be programmed so as to present a sequence of different filters during the course of inspection of each set of well sites, and the camera output is switched or flagged accordingly, so as to allow the different filters to be linked to the different camera output signals.
Use of an interference filter instead of an absorption filter allows improved discrimination of emission wavelengths and the improved blocking of unwanted wavelengths, whether residual excitation reduction or other emissions arising from the excitation.
The use of an interference filter may however introduce two problems.
Such a filter is quite thick (eg 5-10 mm), which can lead to cross talk between well emissions due to loss of spatial resolution. Furthermore, the central wavelength of the bandpass range shifts with angle of incidence eg at 15° from the normal to the filter, the shift is typically 4 nm. This may be acceptable in many instances but at 30°, the shift is typically 16 nm, which is almost certainly unacceptable.
One solution is to compromise light gather efficiency by using fibre optic bundles having smaller numerical aperture of say 0.6 instead of 1. However this will not limit the angle of extreme rays (sin
−1
0.6=37).
Since it is a primary requirement of systems such as described herein, (and. in our concurrent Application) that fibre optic bundles are used having a high numerical input aperture (ie angular acceptance), the numerical aperture of the output end of the fibre optic bundle will also be high. This results in a large cone of rays emanating from the end of each fibre towards the filter. This is generally incompatible with the input requirements of an interference filter which operate best when input rays have only a small angular spread.
According therefore to a further feature of the present invention, optical means is incorporated which effectively reduces the numerical aperture of the output of the coupling plate as seen by the interference filter.
In a preferred arrangement, optical means is provided which cause the light emitted from the coupling plate to be converted into a parallel beam.
In one arrangement magnifying optical means is provided between the output face of the coupling plate and the interference filter so as to present an enlarged image of the coupling plate output face, to the interference filter.
Typically a separate lens is required for each fibre optic bundle. In such an arrangement a mini-lens or a gradient index (GRIN) lens may be placed at the end of each bundle of optical fibres.
Disadvantages of a lens approach include wavelength dependence of the optics, vignetting, limited acceptance numerical aperture, difficulty of restricting angular range of skew rays or rays coming from the edges of the optical fibres and the need for careful alignment of lenses with the fibres.
Alternatively and more preferably, the fibres making up the bundles may increase in cross-section as between the input and output ends of the plate, so as to present an enlarged image of the well emissions to the interference filter.
According to a preferred feature of the invention, therefore a preferred solution involves the use of a fibre optic coupling plate in which the fibres taper in cross-section from the output end to the input end so that the area of each fibre and therefore each fibre optic bundle in the output face of the plate is greater than that of the particular fibre or fibre optic bundle in the input face of the plate.
Typically the size ratio between the input and output ends of the plate is 15.45.
Typically the diameter of the camera input is 45 mm and a well plate has 96 wel

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