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Description  |
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This invention relates to improvements in photometric instruments, and
their use in methods of optical analysis, and in ancillary devices for use
therewith.
The prior art contains numerous disclosures of analytical devices for
handling and metering small volumes of test samples.
G.B. No. 2 090 659 (Instrumentation Laboratory, Inc.) describes test strips
constructed with a self-filling metering channel and a lip or inlet on
which a sample of more than about 10 microliters of for example whole
blood can be placed, so that (for example) 10 microliters is taken up by
capillary action to react with a reagent carried on a fibrous pad above a
filter layer beneath a transparent window. The result can be viewed by the
unaided eye, e.g. as a colour reaction.
G.B. Nos. 2 036 075 (H. E. Mennier), 1 104 774 (J. P. Gallagher), EP Nos. 0
057 110, 0 034 049, 0 010 456 (Kodak), all describe some other aspect of
the uses of capillary channel or chamber dimensions for handling
biological or test fluids.
G.B. No. 1 530 997 (Monsanto) describes the use of coated optical fibres
which can be used in tests that change the light transmitting capabilities
of the waveguides via reactions. WO No. 81/00912 (Buckles) also describes
fibre-optical devices in which the fibre surface or surrounding modify the
light transmission through the core.
U.S. Pat. No. 3,939,350 describes optical measurement of fluorescent
material bound to the surface of a solid transparent prism by a method
involving a single total internal reflection and interaction of the
evanescent wave at the surface of the prism with the bound material.
EP No. 0 075 353 (Battelle) makes specific reference to the
exponentially-decaying (evanescent) external radiation due to light which
is propagated longitudinally in a fibre, and its interaction with
coatings, and this principle is also taken up in immunoassay test devices
of EP No. 0 103 426 (Block) in which light of fluorescence excitation as
well as emission wavelengths is propagated within a antigen--or
antibody--coated optical fibre or plate contacting a capillary-dimensional
sample liquid volume bounded by a tube or another plate and containing a
fluorescent-tagged binding partner of the material coated on the fibre or
plate.
It is an aim of the present invention to provide instrumental arrangements
by which very small liquid samples can be optically analysed in a
convenient and flexible manner to discriminate sample material which is
bound to a solid surface from sample material that remains free in
solution.
According to the present invention we provide methods and instrumental
arrangements for carrying out immunoassays or other chemical or biological
tests, in the course of which a material with light-absorbing or
fluorescence or luminescence properties becomes bound to the surface of a
transparent solid body (especially from a solution or dispersion
contacting the solid body), for example a prism, sheet or fibre, to a
variable extent depending on the presence or amount of an analyte under
test. In these arrangements the transparent solid body is optically
coupled to a photodetector in such a way that the light path from the
material to the detector passes through the solid body and may be therein
totally internally reflected once or a plurality of times, i.e. the solid
body acts as an optical waveguide. Usually the detector yields an
electrical output signal which is processed and used in per se known
manner not in itself constituting this invention, to yield a signal or
indication representative of a parameter of interest in connection with
the test. The arrangement includes a diaphragm or other limitation of the
angle of view of the detector in order to ensure that substantially only
that light is detected which comes from material which is bound to the
surface of the transparent solid body. The transparent solid body acts as
a waveguide for the light which comes from the surface-bound material, and
which can pass by for example transmission, scattering, reflection,
luminescence, fluorescence, or phosphorescence, (for practical purposes
herein considered as a form of fluorescence). The diaphragm or other
limitation of the angle of view of the detector ensures that light from
otherwise similar material that remains in solution or dispersion and
thereby remains spaced from the surface of the transparent solid body, is
not also detected to an appreciable extent. It is within the scope of the
invention to arrange that the detector receives light preferentially from
the bound material, together with a certain amount of light from the
solution or dispersion, and then to provide a compensatory signal from
another detector arranged to receive light from the two sources in a
different proportion, e.g. substantially all from the solution or
dispersion. The invention also extends to alternative arrangements in
which the restriction on the angle of view of the detector is the inverse
of that already described, namely so that the principal detector receives
light substantially only, or at least preferentially, from the solution or
dispersion.
Accordingly, in one aspect the invention comprises a method of optical
analysis of a test sample which comprises a sample material with
light-absorbing or fluorescent, phosphorescent or luminescent properties,
which sample is partly in a liquid phase and partly bound to an adjacent
solid surface, to discriminate the respective parts of said sample
material which are located in the liquid and bound to said solid surface:
comprising the steps of providing as said solid surface a surface of a
transparent solid optical waveguide, and measuring light from the sample
material bound to said solid surface that has passed into and through said
transparent solid optical waveguide with total internal reflections and
emerged from said waveguide at an angle that deviates from the optical
axis of said waveguide by an angle appreciably less than .alpha. where
.alpha.=arcsin.sqroot.(n.sub.2.sup.2 -n.sub.1.sup.2)
where n.sub.2 is the refractive index of the material of the waveguide and
n.sub.1 is the refractive index of the adjacent liquid (generally less
than n.sub.2), and excluding from said measurement substantially all light
that has emerged from said waveguide at an angle that deviates from said
optical axis by .alpha. or more.
In another aspect the invention comprises a combination of a photometric
instrument and a test object adapted therefor, suitable for example for
carrying out immunoassays or other chemical or biological tests,
comprising
(i) a test object such as a slide or cell received and located at a test
object location, for containing a sample material with light-absorbing or
fluorescence or luminescence properties, said test object comprising a
transparent solid body, e.g. a prism, sheet or fibre, to act as a
waveguide, and to the surface of which the sample material can become
bound from a solution or liquid dispersion contacting the solid body;
(ii) a photodetector arranged so that when a test object is at the test
object location (the "in use" condition) the transparent solid body is
optically coupled to the photodetector, so that light propagating in the
transparent solid body can be received by the photodetector after its
emergence;
(iii) (optionally) a light source arranged so that in the "in use"
condition it can illuminate the test material with light (for example at
some transverse angle to the principal direction of propagation of the
light in the waveguide, eg. at or about 90.degree.), so that light from
the test material passes into and through the solid body, via total
internal reflections, to the detector; and
(iv) a diaphragm or other limitation of the angle of view of the detector
in order to discriminate that light which comes from material which is
bound to the surface of the waveguide, e.g. either by transmission,
scattering, reflection, luminescence or fluorescence, from light from
otherwise similar material that remains in solution or dispersion and
thereby remains spaced from the surface of the transparent body, said
limitation of the angle of view corresponding to an angle .alpha. as
defined above.
The invention is considered to rely on the difference in the angular range
of directions of propagation within which the light emerging from the
solid body is distributed, depending on whether the light comes from the
liquid or from a very thin surface bound layer. The solid body can be an
optical fibre or sheet such as a flat solid slide, of glass, silica,
inorganic crystal (eg. sapphire) or plastics material (eg. acrylic
plastics polycarbonate, or polystyrene), and in that case, light from
material in an adjacent liquid of relatively low refractive index, e.g.
close to that of water, emerges at rather large angles off the axis of the
fibre or plane of the sheet. As the refractive index of the bound layer
from which the light comes is the larger and closer to the refractive
index of the solid body, the larger is the angular range of distribution
of the light: this range now includes angles closer to the axis of the
fibre or plane of the sheet. Furthermore, fluorescent or luminescent
molecules or scattering molecules or particles closely adherent to the
surface of the transparent solid can emit light into an angular range
(corresponding to the guided modes of the substrate) part of which would
be a "forbidden" range in the context of ray optics, into which only an
evanescent wave extends and not the main propagated light from sources
located in the body of the liquid, e.g. aqueous liquid. This can result in
light that emerges from the fibre or sheet at exit angles relatively close
to the axis.
We find that where a solid slide or other waveguide is in contact with an
adjacent aqueous liquid containing a fluorescent solute, and fluorescence
is excited by a light source transverse to the axis of the waveguide or
plane of the slide, then the fluorescence emerging from the end of the
waveguide or slide exits mainly at angles of at least about
.alpha.=arcsin.sqroot.(n.sub.2.sup.2 -n.sub.1.sup.2) where n.sub.2
represents the refractive index of the waveguide or slide and n.sub.1
represents the refractive index of the liquid. For acrylic plastics
material, water, and yellow-green fluorescence at 510 nm, .alpha. can be
about 41.degree.. Thus most of the light will emerge at angles off-axis by
41.degree.-90.degree. in either sense and relatively little at angles less
than about 40.degree. in either sense.
In the presence of an adsorbed fluorescent layer at the surface of the
slide or other waveguide, more of the fluorescent light emerging from the
end of the slide or waveguide will emerge at angles closer to the axis.
However, rather little difference is found in the amount of light emerging
within a few degrees, about 5.degree. in either direction, of the axis
itself.
In the case of a glass slide or waveguide and an aqueous solution, and
fluorescent light of 510 nm wavelength, the corresponding angle .alpha.
falls at about 47.degree. off-axis, so that if desired light from
dissolved fluorophor can be detected conveniently in the range above about
49.degree.-52.degree. and up to 90.degree. in either direction off the
axis. Light from an adsorbed layer can be distinguished by its emergence
over a wider range of exit angles including smaller angles less than about
47.degree.. Thus it can be detected conveniently at angles in the range
5.degree.-45.degree. in either direction off the axis.
A low background light level may be detected to a certain extent over a
wide angular range, especially close to the axis and up to about 5.degree.
off axis in either direction.
The effects of background light in the instrumental arrangements described
herein can be dealt with in any of a number of ways. Background light is
found to arise from stray exogenous light, and in the case of examples of
the invention that use a light source to excite luminescence, eg.
fluorescence, it can happen that scattered source light and background
fluorescence from materials other than the wanted sample materials may be
liable to find their way to the detectors. Examples of suitable filter
arrangements to exclude light of other than the wanted wavelengths are
illustrated below.
Background light in the region within the range of .+-..alpha. on either
side of the axis can be detected for example (a) by providing a separate
detector to capture only light emerging along the axis or within a narrow
angular range eg. .+-.5.degree. on either side of the axis and to use the
output of such a detector as an index of background light, (b) to measure
the light emerging within the angular range up to not more than
.+-..alpha., (usually a few degrees less, as described above,) at the
wavelengths of the light source, and to use this measure as an index of
the background component of the light emerging within a similar angular
range at wavelengths to which the principal detector arrangement is
sensitive. Simpler measures are usually enough in the case of arrangements
for detecting bioluminescent or chemiluminescent emission, since a source
of excitation light is absent: a light-tight optical enclosure is
generally enough to deep the background down.
Suitable diaphragms can be arranged as may be desired to select emergent
light of any angle of emergence or range of angles of emergence. In the
case of a circular-section glass or plastics waveguide surrounded by
liquid the diaphragms can be circular or annular diaphragms; and in the
case of a waveguide in the form of a flat slide the diaphragms can be of
rectangular or slit form.
A preferred form of the invention is one in which a fluorescent material in
a liquid adjacent to a transparent waveguide such as a slide or fibre of
for example glass or plastics is excited by light from a light source
transverse to the axis or direction of propagation of light in the
waveguide: light emerging from the end of the waveguide passes through a
diaphragm or other restriction of the angle of view of a subsequent
photodetector, by which the light emerging at angles less than .alpha. may
be distinguished from the light emerging at angles greater than .alpha..
The aqueous liquid in this case can contain reagents and the surface of the
waveguide can have a sensitisation such as a ligand-binding eg.
immunosorbent sensitisation, which together correspond to any known
suitable test such as a ligand binding assay, eg. immunoassay, that
results in a variable degree of deposition or binding of fluorescent
material to the sensitised surface, depending on the presence or quantity
of an analyte under test. In this way the optical arrangements described
herein can distinguish the degree of deposition or binding without the
need for manipulations leading to physical separation of the free and
bound material.
An alternative form of the invention, more suitable for colorimetric than
fluorimetric or luminescence measurements, employs a light source that
launches light longitudinally into a waveguide such as a fibre or slide of
for example glass or plastics material, with a wide range of angles of
incidence, e.g. through a diffuser, or alternatively with a selected
narrower range of angles of incidence chosen to co-operate with the
light-absorbing arrangements described next below: adjacent to the
waveguide is a liquid containing light-absorbing material and reagents by
which an amount of the light-absorbing material can become bound or
deposited on the surface of the waveguide. In the presence of binding,
attenuation occurs particularly of the light travelling in the waveguide
with angles corresponding to emergence angles less than .alpha.. The
absorbing substances in the body of the liquid exert their attenuating
effects on light that emerges at angles greater than .alpha.. Suitable
arrangements of diaphragms or restrictions on the angle of view of a
photodetector arranged to detect the emergent light can then be provided
to discriminate the light affected by the attenuation.
This arrangement has the aim of achieving an optical distinction between
free and bound portions of a material that becomes partly bound to a solid
phase, as is normally the case with many varieties of immunosorbent assay.
But the present method achieves this distinction without requiring an
actual physical separation of the liquid and solid phases (which are of
lower and higher refractive index respectively) such as is normally
required in an immunosorbent assay.
The arrangements described herein are suitable to be applied to bound
sample material which interacts with light in any of a number of ways,
especially for example by absorbance, scattering, fluorescent or
phosphorescent emission, or chemiluminescent or bioluminescent emission.
The chemical features of analytical test methods that exploit these
interactions are known in the art and per se do not constitute this
invention.
The invention is further illustrated by way of example by the following
description and accompanying drawings.
FIG. 1 of the drawings diagrammatically shows optical arrangements of one
embodiment of the invention. FIG. 2 shows in diagrammatic graphical form
the variation of light output with exit angle in an arrangement such as
that of FIG. 1. FIG. 3 gives a block diagram of signal processing
arrangements in an optical instrument as in FIG. 1. FIGS. 4, 4a and 4b
show in diagrammatic scheme alternative optical arrangements usable
according to the invention.
FIG. 1 shows, in fragmentary schematic cross-section, an arrangement
according to an embodiment of the invention, for carrying out optical
analysis of a specific binding reaction, e.g. an immunosorption reaction,
taking place in a layer of aqueous sample liquid 1, by which a component,
e.g. a fluorescent component with optical properties corresponding to
fluorescein, of the material dissolved or dispersed in the liquid becomes
partly bound in a layer 2 (shown with much exaggerated thickness in the
drawing) at the surface of a solid transparent glass, silica or plastics
slide or plate 3. Liquid 1 is located between plate 3 and a second
parallel cover plate 4, which in the example illustrated is transparent
but may be opaque with other instrument configurations. Plates 3 and 4 can
form an integrated sample carrier, e.g. as described in our copending UK
patent application No. 8415018, and copending application of June 13,
1985. The system is illuminated by a light source indicated schematically
at 5 and comprises a flashlamp e.g. as often used in photoflash or
stroboscopic flash units 6, and a filter 7 for selective transmission of
light of excitation wavelength, to give a light output 9 illuminating the
surface of transparent plate 4 and thereby also the liquid layer 1 and
bound material 2. It can be preferable to space the lamp and filter as
close as possible to the sample carrier so long as stray light paths from
source to detector are guarded against.
In an alternative arrangement the plates and liquid and bound layers can be
inverted relative to what is shown in FIG. 1 so that the light 9 impinges
first on the equivalent of plate 3, then layer 2 and liquid 1. A mirror
may be placed on the side of the sample cell opposite to the light source
to increase the effective illumination.
In other alternative arrangements the light source shown can be replaced by
any convenient, e.g. collimated and filtered, light source, or by a laser.
Alternative possible forms of light source can give a.c.-modulated light,
e.g. chopped or pulsed light, as can be provided either by electronic
control or by a segmented rotating chopper vane, according to component
techniques well known in themselves.
Plate 3 is optically clear transparent with parallel plane faces and it has
an end 10 which is substantially smooth and perpendicular to the long
dimensions. The other edges can be made roughened and blackened to remove
stray light.
Light from the liquid 1 and bound layer 2 travels (in part) along the
length of plate 3 undergoing multiple reflections on the way, and emerges
at end 10.
A photodetector 11 is arranged to receive and sense light propagating in
plate 10 as totally internally reflected light, as in the equivalent of an
optical fibre or waveguide, and emerging from end 10, but only receives
light having an exit angle which diverges by not more than an angle
.alpha. as indicated in FIG. 1, measured relative to an axis direction
parallel to the long dimension of plate 3. In other words, light that
diverges from the axis at an angle greater than .alpha. is not to be
caught by detector 11. This is achieved by screening arrangements
including apertured diaphragm 12, placed far enough away from end 10 of
plate 3 so that it makes a practically inappreciable difference
(.ltoreq.about 2.degree.-3.degree.) to the angle whether the light emerges
from the top or bottom of end 10. It can be useful to associate the
apertured diaphragm with a convex lens, eg. a cylindrical lens, to collect
the light exiting over the desired angular range on to the photodetector.
The angle .alpha. is chosen in such a way that it is less than the value
arcsin.sqroot.(n.sub.2.sup.2 -n.sub.1.sup.2)
by an amount sufficient to exclude practically all the light that arises
from the liquid 1 rather than from the layer 2. In the above
trigonometrical formula n.sub.1 represents the refractive index of the
liquid 1 and n.sub.2 represents the refractive index of the solid
substrate, i.e. the plate 3. For the case of fluorescent yellow-green
light around 510 nm and a substrate 3 of acrylic plastics material,
(n.sub.2 about 1.495), used in connexion with an aqueous solution, the
value of the formula is about 41.degree., and we find that a suitable
corresponding value for .alpha. is in practice about >4.degree. less than
this value, e.g. in this case about 37.degree. or less, preferably only a
little less. It is not desirable to set .alpha. very much smaller than
necessary because then there is a risk of excluding light arising from
bound layers which have a refractive index not much greater than that of
the surrounding liquid, or are slightly spaced off (on a molecular scale)
from the surface.
In this way detector 11 receives light preferably from the material bound
to the waveguide surface, though some of the light from this material is
of course lost in other directions.
Additional detectors are in this example provided as follows: detector 14
receives light through a further diaphragm 13, limited to receive light
exiting from perpendicular, optically flat end 10 of plate 3 in an angular
range outside the angular range to which detector 11 is limited, and a
reference detector 15 to receive light in an intensity related to the
effective illumination of the test sample materials by light source 6. A
filter is shown in the drawing between the light exit end of the waveguide
and the principal detector system, to block light of the excitation
wavelength, in manner known per se.
For certain applications it may be desirable to arrange alternative
diaphragms in place of those shown in the drawing, so that for example the
range of angles of view of detector 11 has not only an upper limit .alpha.
but also a lower limit .alpha..sub.1 >0 provided by an additional
diaphragm or screen, two apertures possibly being provided to give
detection of light emerging within the ranges .+-.(.alpha. to
.alpha..sub.1) on either side of the axis direction. In certain
embodiments it is possible to dispense with the equivalent of detector 14
and/or detector 15.
A very limited range of angles of view, e.g. of the form .+-.(.alpha. to
.alpha..sub.1), may be appropriate to increase the effective signal to
noise ratio in cases where the layer to be optically investigated is of
known and well-defined refractive index, or where the angular distribution
of the light from surface-bound material is modified to lie in a narrow
range of angles, eg. by including a 50 nm silver layer showing surface
plasmon resonance on the surface of plate 3 beneath the layer 2: as
described by R. E. Benner et al, J.Phys.Chem. 84 (1980), pp 1602-1606.
FIG. 2 of the accompanying drawings shows in diagrammatic graphical form
the variation of light output with exit angle in an arrangement such as
that of FIG. 1. The horizontal axis of FIG. 2 represents exit angle with
reference to the axis (horizontal in FIG. 1) of slide or plate 3, ie. zero
angle corresponds to light output along the dotted axis of slide or plate
3 in FIG. 1. The vertical axis represents relative fluorescence intensity,
and the several curves of FIG. 2 represent different conditions in the
liquid adjacent to slide or plate 3, as follows. Curve 21 shows the
dependence of fluorescein (0.5 microgram/ml in water in a 20 micron thick
cell space) fluorescence intensity on exit angle, where the fluorescent
material is entirely in solution adjacent slide or plate 3. It is apparent
that very little fluorescent light exits at angles less than about
45.degree.. Curve 22 shows background signal in one experiment due to
binding of bovine serum albumin to slide 3, and curve 23 shows the results
of binding fluorescein-labelled protein to slide 3: the fluorescent light
output at exit angles close to zero is at a minimum comparable with the
background level due to a blank slide as shown by curve 24, and there is a
restricted range of exit angle less than about .alpha. in which
significant light emerges under the conditions of surface-bound
fluorescence, although as shown by curve 21 practically no fluorescent
light output above background exits in this angular range from the
fluorescent substances in solution.
Thus the signal from detector 11 in FIG. 1 can discriminate fluorescence
due to surface-bound fluorophor in layer 2 from fluorescence due to
fluorophor in the body of liquid 1. The invention is not limited to
fluorophors or to fluorescein and its conjugates. Fluorescein is however a
particularly convenient fluorophor for use in this invention, as also is
rhodamine, eg. used with excitation wavelength 550 nm and emission
wavelength 590 nm.
In an alternative embodiment briefly mentioned above, detector 11 as in
FIG. 1 can receive the light from a diaphragm with both upper and lower
angular limits of its aperture: in this case the light that exits from
slide 3 very close to its axis, eg. within about 5 degrees of the axis, is
not caught be detector 11. If desired a separate detector of this light
can be installed, so that a background level control signal can be
derived, and included in the signal processing.
Not shown in the drawings because they constitute per se-conventional
structural features, are positive-registration arrangements to mount plate
3 in predetermined alignment relative to the rest of the optical system,
overall screening, covering and optical absorbing features to keep out
stray light during the measurements, and mechanical means, if required to
shift the optics and sample(s) relative to each other, to enable
successive measurement of a plurality of samples.
Also not shown in detail are the electrical circuitry and modules, composed
of per se conventional units, for taking and processing the signals
arising from the detectors 11, 14 and 15 shown in FIG. 1. A schematic
block diagram of these arrangements is given in FIG. 3. In one suitable
arrangement, the light source 6 of FIG. 1 is a flashlamp 31 (FIG. 3)
controlled by controller/power-supply 30: flashlamp 31 illuminates sample
cell 4 of FIG. 1 (shown as 32 in FIG. 3).
The light incident on the detectors (11, 14 and 15 in FIG. 1-33, 34 and 35
in FIG. 3) gives rise to electrical signals that are then processed by
signal-processing circuitry. This circuitry incorporates any of several
known photoelectric signal processing techniques and in particular can
incorporate a synchronous lock-in or boxcar detection arrangement actuated
by additional signals derived from the reference detector to synchronise
with the chopping frequency of the light source. The signals from the
detectors are then processed, e.g. by a.c. and d.c. separation,
sample-and-hold stages, analogue-to-digital converters, or as may be
appropriate, and combined, e.g. by linear combination or ratio circuitry
or digital processing, to obtain processed e.g. normalised data signals,
or output signals directly representative of any convenient desired
parameter. A signal or data output is taken in any desired form, e.g.
analogue, digital, graphical, printed or electronically stored or
processed data, by the use of appropriate known output or interfacing
circuitry and apparatus. Especially, any suitable form of normalisation of
the signals from detectors 11 and 14 by reference to detector 15 output
can be provided for, and it may be desirable to form combinations, e.g.
linear combination or ratios, of the signals from detectors 11 and 14 to
give better effective discrimination between the different light sources
of interest, subtraction of background light, and/or rejection of changes
in the absolute intensity of the pump light. If desired, the circuitry can
provide for the making and signal-processing of a succession of light
measurements at determined time intervals, to allow the kinetics of the
(eg. binding) reactions in the sample cell to be detected or measured.
In the particular arrangement shown schematically in FIG. 3, detector 35
provides timing signals via reset 36 for integrating amplifiers 37 and 38,
and gives rise to a delayed trigger signal by delay unit 39 and trigger 40
to control a/d converter 41 which provides a digital output based on the
output of analogue divider 42. Typically a suitable ratio signal to be
delivered by divider 42 is representative of the ratio of outputs of
integrators 37 and 38.
Integrating amplifier 37 thus provides a signal representing the output of
the detector of light that exits from waveguide 3 at angles less than
.alpha., i.e. it is responsive to light from the adsorbed part of the
sample material, and integrating amplifier 38 provides a signal
representing the output of the detector light that exits from waveguide 3
at angles greater than .alpha., i.e. it is responsive to fluorescence from
the solution as well as the adsorbed sample material.
This photometer arrangement is provided with all usual suitable screening
and cover arrangements to minimise stray light and electrical
interference, and suitable holders and registration devices to hold the
plate arrangements in predetermined orientation relative to the optical
system of the photometer, as well as any desired electronic calibration
and stabilisation arrangements to prevent the effects of instrument drift
from disturbing the data output in uncontrolled fashion. The sample holder
may be present in multiple and movable form and/or the optical system may
be movable to subject more than one sample slide to measurement
successively.
Where the light source is a flash light-source, it can be useful in certain
applications to include arrangements known in themselves for gating the
photodetector response open, especially a few microseconds after the flash
in order to allow background fluorescence to decay, e.g. as mentioned in
Specifications EP No. 0 104 926 and U.S. Pat. No. 4,341,957.
FIG. 4 shows in diagrammatic scheme an optical arrangement for an
instrument as in FIG. 1 but with the following modifications. (Like
numerals in FIGS. 1 and 4 indicate corresponding parts.) Flash light
source 6 is provided with a collimating system 41 to minimise stray light
throughput to detectors 11 and 14. A reflective surface 42 is provided
opposite the light source to increase the level of illumination on the
sample. Detector 15 is shown but may if desired be omitted in this
embodiment as alternative means of compensating for excitation light
intensity variation and background light is provided by beamsplitter cube
43, detector 44 and the positions of filters 45 and 46 to exclude light of
excitation wavelength (as compared with the single filter adjacent
waveguide light exit surface 10 in FIG. 1). The light path to detector 44
is transmissive to light of the excitation wavelength and accordingly the
signal detector 44 largely represents the intensity of background light
exiting from face 10 of the waveguide within the angular range
.+-..alpha.. Filter 45 excludes as much as possible of this light from
principal detector 11, and the residual background component of the signal
from detector 11 can be largely compensated by a signal derived from
detector 44. Detector 14, provided with filter 46 to exclude light of
excitation wavelength, can be used to provide a signal to normalise the
output from detector 11.
FIGS. 4a and 4b schematically show alternative ways of illuminating the
sample cell. In FIG. 4a, light source 6 is surrounded by filter 7 to
select light of excitation wavelength, and an elliptical (cylindrical)
reflector 47 of which lamp 6 occupies one focus and the sample cell the
other focus, to give increased illumination intensity on the sample. In
FIG. 4b, light from the source 6 illuminates the sample layer 2
substantially in the plane of layer 2 but substantially transversely of
the main direction of propagation of light in the waveguide to the
principal detector system (shown here in schematic outline at 48).
It is within the scope of the invention to include as part of layer 2
(FIGS. 1 and 4) in the sample cell a thin metal layer (eg. a silver metal
layer about 50 nm thick), formed on the surface of the waveguide 3, and on
the surface of which a layer of bound sample material is formed in known
manner, eg. as an immunosorbent with attached fluorescently-labelled
ligand. This enables the use, in conjunction with the instrumental
arrangements described herein, of the per se known phenomenon of surface
plasmon resonance (s.p.r.), as described in B. Liedberg et al, Sensors and
Actuators, 4 (1983) 299-304, or by Benner et al, cited above, to provide
discrimination of sample material bound to layer 2 by means of its effect
on the resonant angle at which light energy is maximally dissipated or
coupled instead of propagating and exiting through end 10 of waveguide 3.
For this purpose, diaphragm 12 can for example be adjusted to a narrow
slit width corresponding to an angle near the angle of minimum
transmission or coupling due to s.p.r. dissipation or coupling, and
detector 11 can then be used to detect changes in light output due to the
per se known changes that occur in the s.p.r. angle of maximal dissipation
or coupling when sample material becomes bound to layer 2.
This invention is susceptible to any of several variations and
modifications within its scope, and extends to the use of any one or more
of the singular and several features of the foregoing description and
accompanying drawings.
It is also to be noted that many of the devices made and described in our
copending application no. 06/834247, claiming priority from our patent
application filed on 13th June 1984, G.B. No. 84 15018, and entitled
"Devices for Use in Chemical Test Procedures", the entire disclosure of
which is hereby incorporated by reference into this present specification,
can be usefully applied to carry the test samples to be measured optically
by the photometric methods and arrangements described hereinabove.
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