WO2012070942A1 - Method and apparatus for surface plasmon resonance angle scanning - Google Patents

Method and apparatus for surface plasmon resonance angle scanning Download PDF

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Publication number
WO2012070942A1
WO2012070942A1 PCT/NL2011/050806 NL2011050806W WO2012070942A1 WO 2012070942 A1 WO2012070942 A1 WO 2012070942A1 NL 2011050806 W NL2011050806 W NL 2011050806W WO 2012070942 A1 WO2012070942 A1 WO 2012070942A1
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Prior art keywords
element
optical
apparatus
optical element
light
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PCT/NL2011/050806
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French (fr)
Inventor
Richardus Bernardus Maria Schasfoort
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Ibis Technologies B.V.
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Priority to NL2005761 priority Critical
Priority to NL2005761 priority
Priority to NL2005902A priority patent/NL2005902C2/en
Priority to NL2005902 priority
Application filed by Ibis Technologies B.V. filed Critical Ibis Technologies B.V.
Publication of WO2012070942A1 publication Critical patent/WO2012070942A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55Specular reflectivity
    • G01N21/552Attenuated total reflection
    • G01N21/553Attenuated total reflection and using surface plasmons

Abstract

The invention relates to an apparatus for surface plasmon resonance (SPR) scanning, the apparatus comprising: - a sensor assembly comprising a first optical hemispherical element and an SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical hemispherical element; - an illumination assembly comprising a light source and a second optical element; - a detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element; wherein the detector assembly is configured to adapt the position of at least one of the sensing element and third element during scanning to focus the reflected light in a focal point located at both the first focal plane of the first element of the sensor assembly and the third focal plane of the third element of the detector assembly.

Description

METHOD AND APPARATUS FOR SURFACE PLASMON

RESONANCE ANGLE SCANNING

The invention relates to an apparatus for surface plasmon resonance scanning, the apparatus comprising a sensor assembly, an illumination assembly and a detector assembly. The invention also relates to a method of surface plasmon resonance scanning.

Surface plasmon resonance (SPR) scanning is a well known optical technique for determining the adsorption of material onto planar conductive (metal) surfaces or onto the surface of conductive nanoparticles by measuring changes in refractive index near a sensor surface exposed to the material. The technique involves illuminating the conductive surface or the conductive nanoparticles of a sensor element with p-polarized light . Photons of the p-polarized light reflected against a thin conductive layer interact with free electrons in the conductive layer, inducing a wave-like oscillation of the free electrons, thereby reducing the reflected light intensity. This phenomenon yields a maximum effect at a typical angle, the so-called SPR angle.

Changes in the refractive index near the sensor surface result in a shift of the SPR angle. Classical SPR imaging instruments use reflectivity as parameter for measuring refractive index changes. Surface plasmon resonance angle scanning relies on measuring changes in refractive index by determining changes in the SPR angle. The majority of the SPR systems are of the convergent beam type. In other types of SPR systems use is made of a more or less parallel beam to image the sensor surface on a sensor.

Some of the systems used for SPR imaging in the prior art represent fixed-angle instruments. These instruments are based on an almost linear relationship between the change in reflected light intensity and the mass of bound analyte . This means that a fixed incident light angle is employed and mass changes are estimated from the intensity of the reflected light.

Drawback of these fixed-angle instruments is that the range where the linear relationship between the reflected light intensity and the mass of bound analyte applies, is limited. Furthermore, the optimal angles differ considerably when ligand or analyte panels that have different molecular weights are to be monitored. Immobilized ligands of different size and or different density will induce an initial shift. Monitoring multiple biomolecular interactions in parallel on a micro-array may lead to monitoring at non-optimal angles.

A more reliable parameter that reflects mass changes on an SPR sensor surface is the SPR angle shift. The SPR angle shift may be determined by a technigue called incident angle scanning. A range of angles is scanned by stepping through the range and measuring the reflected light intensity at each of the angles. The measured reflected light intensity is plotted against the angle of incident. From these reflectivity versus angle plots, an SPR curve is constructed by curve fitting. The SPR angle can be determined from the SPR curve.

It is to be noted that the word scanning can apply to two different notions in this document. In its primary meaning, scanning relates to angle scanning, this means lighting a surface seguentially under a number of different angles of incident, for instance by changing (moving) the optical axis or by using a tiltable mirror. Secondary, scanning can mean using a narrow beam to light different portions of the surface seguentially, irrespective of the angle of incident. Furthermore, if in the present document reference is made to a change, variation or adaption of the position of an optical element then this reference is not to be construed as being limited to a change, variation or adaption of the location of the optical element. It also may involve a change, variation or adaption of the orientation of the optical element . Scanning the sensor surface involves imaging the surface on a sensing element, such as a camera. In the current measurement setup the light distribution of the image provided on the sensing element is not uniform. Sometimes a relatively high intensity spot is formed at a position corresponding to the centre of the sensor surface while in other parts of the image the sensed light intensity is relatively low. A non-uniform illumination of the sensing element may be detrimental to the scanning results.

In situations in which the sensor surface has multiple spots or regions of interest (ROI's), for instance when the sensor surface is provided with a 100 spots microarray, the results of the measurements obtained from individual ROI's may give a baseline variation between different ROI's. ROI's closer to the centre of the image surface display the highest and the lowest signals, with a variation of up to about 50% between the different ROI's. This baseline variation has a negative impact on the performance of the scanning method.

A further drawback of the known scanning methods and apparatus is that the position of the image of the ROI's on the sensing element may vary depending on the angle of incidence of the light impinging on the sensor surface. The position variation is sometimes referred to as the walking spot effect. It is clear that this effect may impede the correct and efficient scanning operation by the apparatus .

NL 1 022 916 is an apparatus for Surface Plasmon Resonance (SPR) scanning is disclosed. The locations of the tiltable mirror at the illumination side and the detector at the detection side of this apparatus may be changed by moving both the mirror and the detector in a direction perpendicular to the sensor surface of the hemispherical prism. However, also this known apparatus may still suffer from the presence of relatively high intensity hot spots and/or walking spots, thereby reducing the guality of the scanning operation. The object of the present invention is to provide an SPR scanning apparatus and method wherein at least some of the drawbacks of the prior art have been removed or at least reduced.

A further object of the present invention is to provide an SPR scanning apparatus and method that are more efficient and/or more accurate with respect to existing apparatus and method.

A further object of the present invention is to provide an SPR scanning apparatus and method wherein the effect of walking spots during scanning, especially during imaging small objects e.g. living cells, is at least partly reduced.

According to a first aspect of the invention at least one of the objects is achieved in an apparatus for surface plasmon resonance (SPR) scanning, the apparatus comprising:

- a sensor assembly comprising:

- an essentially hemispherical first optical element, substantially transparent to light and defining a first optical element focal plane and a sensor surface;

- an SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical element;

- an illumination assembly arranged to direct light diverging from an illumination assembly focal point to the sensor surface of the first optical element, the light arriving at the sensor surface at varying angles of incidence as a substantially collimated light beam;

- a detector assembly for the spatially resolved acguisition of a light property, for instance the intensity, of light reflected at the sensor surface of the first optical element at varying angles of incidence, the detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element having a third optical element focal plane;

wherein the detector assembly is configured to adapt the location and orientation of at least one of the sensing element and third optical element to focus the reflected light in a focal point located at both the first focal plane of the first optical element of the sensor assembly and the third focal plane of the third optical element of the detector assembly.

In a preferred embodiment the location and orientation of both the sensing element and the third optical element may be adapted to even better focus the reflected light in the focal point that is located at the first and third focal plane.

It is to be noted that angle scanning of the sensor surface may include an angle off-setting phase wherein the sensor surface is scanned with a relatively large angle step size (for instance providing a resolution of about 100 millidegrees) and the actual scanning phase wherein the sensor surface is scanned with a relatively small angle step size (for instance providing a resolution of about 0,1 millidegrees) . The adaption of the position of the focal points of the illumination assembly and the detector assembly as described herein can be employed during the angle off-setting phase, the scanning phase or during both the angle off-setting and scanning phase. The first optical element may be a generally hemispheric prism or similar element, such as a prism in combination with two spherical lenses. The third optical element may be formed by one or more spherical lenses as well, but other types of optical elements may be used instead. The sensing element may be an electronic camera, for instance a CCD camera, a CMOS camera or a similar device. The light source may be a p-polarized narrow-band light source. Other types of light sources are possible as well. The light source may also be a halogen white light source with bandpass filter, a light emitting diode (LED) or superluminescent light emitting diodes (SLED) . A laser source can also be used, preferably with an incoherent processed light beam.

At the side of the detector assembly, the point in which the light reflected from the sensor surface is focussed generally changes position during the scanning operation, i.e. during the illumination of the first optical element at varying angles of incidence. As a result of the movement of this so-called focal point, the optimal conditions for projecting the reflected light onto the sensing element cannot always be met in stationary detection assemblies. According to embodiments of the present invention, however, the position of the sensing element, the third optical element or both the sensing element and the third optical element may be adapted depending on the position change of the focal point. For instance, the elements may be moved in such a way that the focal point is at the intersection of the focal planes of the first and third optical elements. The collimation of the light reaching the sensing element may thus be better maintained during the scanning process.

In embodiments of the invention the detector assembly comprises an orientation adjustment unit for adjusting the orientation of the sensing element and/or the third optical element with respect to the first optical element. This unit makes it possible to optically (fine) tune the detector assembly by suitable adapting the orientation of the sensing element and/or the third element. The adjustment unit may be constructed to allow the orientation of the elements to be adapted manually. In other embodiments the adjustment unit comprises drive elements, such as electric motors or similar elements, to automatically adjust the orientation of the optical element/sensing element of the detector assembly, for instance based on a signal received from a manual controller (for instance a joystick or a similar device) and/or a signal received from the sensing element . The signal from the sensing element is representative of the image sensed by the sensing element (camera) and the drive elements can therefore be controlled based on the image sensed by the sensing element.

In order to correct for perspective distortion the third optical element, the sensing element and the first optical element may be essentially maintained in Scheimpflug condition. More general, the first optical element and the third optical element, or the first optical element and the sensing element, or the first optical element, the third optical element and the sensing element may be positioned (in the sense of location and orientation) such that they are essentially oriented in Scheimpflug position. Maintaining the elements in Scheimpflug condition has a generally positive effect on the scanning results .

In an embodiment of the present invention the apparatus comprises a frame having a tiltable frame element at which the sensing element and/or the third optical element are attached. The sensing element and third optical element may be attached to the same tiltable frame element so that tilting the frame element results in a tilting movement of the optical element. The sensing element and third optical element may also be attached to separate tiltable frame elements. This makes it possible to move the sensing element and third optical element independently.

The tiltable frame element may be configured to move the sensing element and/or third optical element in the plane of incidence so that the sensing element and/or the third optical element may be tilted to an optimal position for imaging the sensor surface on the sensing element. For instance, the sensing element may be positioned on a pivot arm construction to allow optical axis scanning.

At the side of the illumination assembly a similar configuration may be applied. According to another aspect an apparatus for surface plasmon resonance (SPR) scanning is provided, the apparatus comprising:

- a sensor assembly comprising:

an essentially hemispherical first optical element, substantially transparent to light and defining a first optical element focal plane and a sensor surface;

- an SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical element;

- an illumination assembly arranged to direct light diverging from an illumination assembly focal point to the sensor surface of the first optical element, the light arriving at the sensor surface at varying angles of incidence as a substantially collimated light beam;

- a detector assembly for the spatially resolved acguisition of a light property, for instance the intensity, of light reflected at the sensor surface of the first optical element at varying angles of incidence, the detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element having a third optical element focal plane;

wherein the illumination assembly is configured to move the position of the illumination assembly focal point during scanning of the sensor surface at varying angles of incidence so as to essentially align this position with the first optical element focal plane .

If the illumination assembly is configured to move the position of the illumination assembly focal point during scanning of the sensor surface at varying angles of incidence such that this position of the focal point is essentially aligned with the first optical element focal plane, then the light entering the first optical element remains substantially parallel (for instance during a stepwise change of the angle of incidence) . This reduces the presence of high intensity spots and the occurrence of the walking spot mentioned earlier.

In embodiments of the invention the illumination assembly comprises a light source and a second optical element defining an illumination assembly focal plane. The second optical element may be a spherical lens or, preferably, an aspherical lens, and the illumination assembly focal plane then is the focal plane of this lens. In other embodiments the illumination assembly may comprise one or more pinholes and/or one or more optical fibre light sources to generate the divergent light beam directed towards the first optical element.

The movement of the illumination assembly focal point, for instance by moving the position of the second optical element (and/or other elements of the illumination assembly), by moving the pinholes/fibres or by selectively switching the pinholes/fibers on and off, may be accomplished by any suitable means. For instance, the second optical element may be attached to a pivot arm. When the movement of the focal point is such that this position of the focal point is essentially aligned with the first optical element focal plane, then the light entering the first optical element remains parallel (during a stepwise change of the angle of incidence) .

It is to be noted that movement of the position of the illumination assembly focal point generally results in a movement of the image on the sensing element. This movement may be at least partly compensated for by a suitable movement of the sensing element, the third optical element or both the sensing element and third optical element. In specific embodiments of the present invention the position of the third optical element and/or the sensing element is changed so as to minimize the movement (walking spot effect) of the image on the sensing element.

In order to better correct for perspective distortion at least some of the elements of the detector assembly need to be essentially maintained in Scheimpflug condition. To the surprise of the inventors the scanning results can be improved, according to a further aspect of the invention, when additionally of alternatively, in a similar manner one or more parts of the illumination assembly are maintained in Scheimpflug condition. For instance, if the light source of the illumination assembly comprises a tiltable mirror for controlling the angle of incidence, the tiltable mirror, the second optical element and the first optical element may be arranged to essentially maintain the Scheimpflug condition. This can be done in a pre-scanning phase (e.g. a calibration stage) before the actual measurements. In other embodiments this is accomplished during the scanning process, i.e. during varying the angle of incidence by variation of the tilt angle of the mirror.

In a further preferred embodiment both the illumination assembly and the detector assembly are arranged according to the Scheimpflug principle so that the elements of the assemblies fulfil the (double) Scheimpflug condition. In a still further embodiment the tiltable mirror/second optical element on the one hand and the camera/third optical element on the other hand are arranged at mirrored positions (more specifically, mirrored positions and locations (i.e. symmetrically relative to an axis perpendicular to the sensor surface of the first optical element ) .

In embodiments of the invention the second and third elements are selected to have the same focal distance. This creates an essentially equal radiation and reflection path and will reduce the walking spot effect mentioned above, when alignment of the optical elements is correct.

In further embodiments of the invention at least a sensing area of the sensing element is moved essentially in synchronisation with the movement of the tiltable mirror.

When the tiltable mirror is moved during the scanning operation to scan the sensor surface at varying angles of incidence, the sensing element, or the sensing area of the sensing element, is moved as well, preferably in synchronisation with the angle movement of the tiltable mirror, so that a suitable positioning of the sensing area may be achieved, even in the scanning phase (i.e. during the actual scanning of the sensor surface at varying angles of incidence) . The movement of the sensing area may be accomplished mechanically (by mechanical means only) or (also) using one or more electric drive motors controlled by control logic.

As mentioned above, it is not necessary of have the entire sensing element following the movement of the tiltable mirror. For instance, in case of a CCD camera, only the light sensitive surface of the camera needs to be repositioned to follow the movement of the tiltable mirror .

According to another aspect of the present invention an apparatus for surface plasmon resonance (SPR) scanning is provided (preferably in accordance with the apparatus described above), the apparatus comprising:

- a sensor assembly comprising:

- an essentially hemispherical first optical element, substantially transparent to light;

- a SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical element;

- an illumination assembly for the illumination of the sensor surface of the first optical element at varying angles of incidence with a substantially collimated light beam,

- a detector assembly for the spatially resolved acguisition of a light property, for instance the intensity, of light reflected at the sensor surface of the first optical element at varying angles of incidence, the detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element; wherein the third optical element, the sensing element and the first optical element are positioned in Scheimpflug position.

For instance, the detector assembly and/or illumination assembly may be arranged so that an imaginary axis extended from the image plane at the sensor surface, an imaginary axis extended from the optical element, orthogonal to its optical axis, and an imaginary axis extended from the sensing element, orthogonal to the optical axis of the sensing element, intersect in essentially one common point (herein also referred to as the (virtual) Scheimpflug point) .

More generally, the Scheimpflug point is the position wherein the imaginary axis extending from the (image plane at the) sensor surface, the imaginary axis extended from the optical element, orthogonal to its optical axis, and the imaginary axis extending from the sensing element, orthogonal to the optical axis of the sensing element, intersect.

In an embodiment of the invention the light source of the illumination assembly comprises a mirror to illuminate the sensor surface, wherein the mirror, the second optical element and the first optical element are positioned in Scheimpflug position. The positioning of one or more of the elements may be accomplished in many ways . In one particularly useful embodiment the apparatus further comprises :

- a support element arranged so as to be movable along a guide extending along an imaginary axis extended from the image plane at the sensor surface,

- a first support arm connected to the sensing element,

- a second support arm connected to the third optical element ;

wherein the first and second support arms are pivotably connected to the support element.

The Scheimpflug point is always on the line extended from the image plane of the sensor surface. Accordingly, the support element is movable along the axis extended from the image plane and it should be possible to find a position along the guide wherein the support elements may bring the sensing element and both optical elements in the Scheimpflug state. A similar construction may be provided at the illumination side of the apparatus. For instance, the apparatus may comprise:

- a support element arranged so as to be movable along a guide generally extending along an imaginary axis extended from the image plane at the sensor surface,

- a first support arm connected to the mirror,

- a second support arm connected to the second optical element ;

wherein the first and second support arms are pivotably connected to the support element.

The arms enable the mirror and the second optical element to be tilted easily. In an embodiment wherein the support arms are pivotable with respect to a common pivot point (and the support element is movable in line with the sensor element), the individual movements of the mirror and second optical element and/or the individual movements of the sensing element and the third optical element may be made to correspond to the displacements needed to keep the illumination assembly and/or the detector assembly in Scheimpflug configuration. For instance, the apparatus may comprise a drive construction for driving the support element to and forth across the guide. The apparatus may further comprise a controller for controlling the drive means, for instance controlling the movement of the support element so that the position of the pivot of the support arms is automatically caused to coincide with the actual position of the Scheimpflug point.

The illumination assembly may further comprise an angle controller for controlling the tilt angle of mirror and or thereby indirectly controlling the angle of incidence of the light beam striking the sensor surface. When the Scheimpflug point moves towards or away from the first optical element, for instance when the angle controller causes the angle of incidence to change, the controller may cause the support element to follow this movement in an attempt to keep the Scheimpflug condition.

In some of the embodiments the second and/or third optical element is a lens. In other embodiments the second and/or third optical elements comprise one or more pinholes, optical fibres or similar optics. In an embodiment of the present invention the second optical element is comprised of an essentially non-transparent component provided with a plurality of pinholes. The pinholes may be displaced to their respective positions so as to set the position of the second element focal point. More specifically, to set the focal point from which the light is directed (in a divergent manner) to the first optical element.

In other embodiments the pinholes may be selectively made transparent or non-transparent to displace the position of the second element focal point. This can be accomplished by arranging light sources, for instance LED's, behind the pinholes and by selectively turning on and off the light sources. In case the first optical element is a hemispherical prism the pinholes may be situated at a virtual hemispherical focus plane relative to the optical centre of the prism. In this way a substantially parallel light beam may be present in the first optical element, for different angles of incidence.

Alternatively or additionally use can be made of optical fibres which are configured to provide for a divergent light beam. In embodiments of the present invention the light source and second optical element are combined into essentially a component comprising a plurality of optical fibres. The optical fibres may be selectively and subseguently switched on and off, for instance using a suitable controller for controlling the fibre, in order to displace the position of the second element focal point.

An advantage of the above-referenced embodiments may be that no moving parts and/or drive means are needed to displace the focal point from which the light propagates in a divergent manner towards the first optical element. Furthermore, the speed of angle scanning may be increased relative to the embodiments using mechanical positioning means (for example means for tilting the tiltable mirror) .

In an embodiment of the invention the SPR sensor element comprises an array of regions of interest (ROI's) to be scanned with an angle of incidence from a range of angles of incidence and to be imaged on the sensing element. For instance, the sensor element may be spotted with one or more different ligands, the spots forming the regions of interest to be scanned.

In an embodiment the apparatus comprises a fluidic system for delivering and exposing a sample medium to at least the sensor element. The fluidic system may comprise one or more flow cells are cuvettes in which the medium to be tested can be accumulated.

In embodiments of the invention the conductive material comprises a metal, for instance silver, gold, copper, titanium or chromium.

In a further embodiment the first optical element and, preferably, the second and third optical elements, are made of glass . In a preferred embodiment the apparatus has the so-called Kretschmann configuration wherein the metal film is evaporated or sputtered onto the glass block. The light is illuminating from the glass and an evanescent wave penetrates through the metal film .

The light source may be a light emitting diode (LED) mounted on an optical fibre acting as a pinhole. Preferably, the inner core of such optical fibre has a diameter of less than 1 mm, even more preferably less than 200 micrometer. In another embodiment the light source comprises a mirror reflecting light from an array of light emitting diodes (LED's).

In an embodiment the first optical hemispherical element is optically matched to a disposable sensor element, preferably the hemispherical lens itself, using an optogel, refractive index matching oil or other means for optical connection to the disposable sensor element.

In operation, the first optical hemispherical element may be horizontally oriented and the sensor element may be arranged at the top side of the first optical hemispherical element. This allows the measurement of gravitational processes of particles in the liguid and to get rid of air bubbles more easily.

In embodiments of the invention the at least one of the orientation adjustment units comprises a drive motor connected to a control module, the control module being connected to the sensing element and being configured to tune the orientation of the sensing element and/or at least one of the optical elements based on a feedback signal received from the sensing element.

In embodiments of the invention the apparatus comprises a control module configured to control the orientation of at least one of the second optical element, the third optical element and the sensing element based on a signal received from the sensing element, for instance a signal representative of the image detected by the sensing element. The orientation of the elements may be controlled by the orientation adjustment units or in similar units. In some embodiments both the orientation and location one or more of the elements may be controlled based on the signal received from the sensing element.

Furthermore, the orientation may be controlled in a coarse tuning phase and/or in a fine tuning phase. In the coarse tuning phase the location of the sensing element, the third optical element and the second optical element may be set, while in the fine tuning phase the location can be set in a relatively more accurate manner. In the fine tuning phase the orientation of the elements may be set as well.

In embodiments of the invention the location and orientation are set in the pre-scanning phase only and remain fixed in the subseguent scanning operation (in the angle offsetting phase and in the actual scanning phase) . In other embodiments the position (i.e. location and/or orientation) of at least one of the elements is set in the angle offsetting phase and the scanning phase as well.

According to another aspect of the invention a method for surface plasmon resonance (SPR) scanning is provided, the method comprising :

- angle scanning a surface of an SPR sensor element provided at the sensor surface of a first optical hemispherical element by illuminating the sensor surface at varying angles of incidence with a substantially collimated light beam;

- measuring a light property, for instance the intensity, of light reflected at the sensor surface by sensing the image of the illuminated sensor surface, received from a third optical element, by a sensing element;

the method further comprising:

- adapting the position of at least one of the sensing element and third element during scanning so as to focus the reflected light in a focal point located at both the first focus plane of the first element of the sensor assembly and the third focal plane of the third element of the detector assembly.

According to another aspect of the invention a method of surface plasmon resonance (SPR) scanning, is provided, the method comprising :

- angle scanning a surface of an SPR sensor element provided at the sensor surface of a first optical hemispherical element by illuminating the sensor surface at varying angles of incidence with a substantially collimated light beam

- measuring a light property, for instance the intensity, of light reflected at the sensor surface by sensing the image of the illuminated sensor surface, received from a third optical element, by a sensing element;

the method further comprising:

- adapting the position of the illumination assembly focal point during scanning of the sensor surface at varying angles of incidence so as to essentially align this position with the first optical hemispherical element focal plane.

Further characteristics, advantages and details of the present invention will become apparent from the following description of several embodiments thereof. Reference is made to the annexed drawings, wherein:

Figure 1A is a schematic view of a part of an embodiment of the SPR angle scanning apparatus wherein the present invention is applied;

Figure IB is enlargement of a part of figure 1A showing the conical and cylindrical light beams directed towards the sensor surface ;

Figure 2 is a side view of the embodiment of figure 1; Figure 3 is a top view of the embodiment of figures 1 and

2;

Figure 4 shows a SPR curves associated with a plurality of regions of interest (ROI's) resulting from a scan in accordance with the present invention;

Figure 5 is a side view of the embodiment of figure 1, wherein the camera and lens are tilted to restore the Scheimpflug condition ;

Figure 6 is a schematic view of a further embodiment of the invention, wherein the illumination assembly focus point is displaced by movement of the combined optical element and light source in order to maintain a parallel light beam inside the first optical hemispherical element during at least a part of the scanning procedure;

Figure 7 is a schematic view of a further embodiment, wherein the illumination assembly focus point is moved by movement of an aperture ;

Figure 8 is a schematic view of a further embodiment of the invention, wherein the displacement of the illumination assembly focus point is realised by controlling a plurality of light sources arranged behind a plurality of apertures; and

Figure 9 is a schematic view of a further embodiment of the present invention, wherein the displacement of the illumination assembly focus point is realized by selectively turning on and off a plurality of optical fibres;

Figure 10 is a schematic view of an example of a mechanical construction that is used to keep the sensing element, preferably also the third optical element, in Scheimpflug arrangement; and Figure 11 is a schematic side view of a scissor-like construction carrying the optical elements in accordance with a further embodiment of the present invention.

An SPR scanning device 1 (figures 1-3), for instance a biosensor for determining information about the kinetics of biomolecular interactions, in the above-mentioned Kretschmann configuration may comprise a sensor assembly 3 interacting with the medium to be scanned, an illumination assembly 9 for illuminating the sensor assembly 3 and a detector assembly 20 for detecting the influence of the biomolecular interactions on the light illuminated on the sensor assembly 3.

The sensor assembly 3 comprises a generally hemispherical prism 4, for instance made of glass or any other suitable optically transparent material . The generally flat upper surface 5 of the glass hemisphere 4 is coated with a thin layer 6 of conductive material, for instance gold. The thickness of the conductive layer typically is approximately 30-60 nm. The thin layer 6 is located between two media with different refractive indices. At the one side, the glass hemisphere 4, and at the other side another media, for example a carrier liguid or a gas. The thin layer 6 forms a sensor element for sensing the interactions of the liguid or gas with one or more regions (spots) on the sensor surface .

The carrier liguid or gas is contained in a fluidics system for back and forth flow of the medium. A fluidics system may comprise one or more flow cells to enable interactions to occur at the surface of the sensor element. Numerous different types of fluidics systems are known, for instance serial flow cell systems, independent flow cell systems and hydrodynamic addressing flow cell system. However, since fluidics systems as such are known in the art, a detailed description thereof is not needed for understanding the present invention.

The refractive index of the hemisphere 4 is reguired to be higher than the refractive index at the other side (sensor surface side) of the conductive layer 6 e.g. water. At the other side of the layer 6 specimens (8) are present that are to be monitored by the SPR process. These specimens (8) may influence the refractive index at this side of the conductive layer 6. The influence on the refractive index can be determined and may characterize the interactions occurring at the sensor surface.

The illumination assembly 9 comprises a narrow band light source, for instance halogen light with a bandpass (BP) filter, an LED with or without BP, a superluminescent LED, a laser made non-coherent by fast rotating transparent medium or any other suitable means that couples light into a light guide 15. The light leaving the light guide 15 is collected by a collimator 11 that collimates the incoming light and projects the collimated light onto a p-polarizer 12. Optionally the light is subjected to a band pass filtering process in a band pass filter (not shown in the drawings) . The narrow band p-polarized (possibly band limited) light is projected on a mirror element 13. The mirror element 13 is pivotably attached to a frame (cf . figure 11) may be tilted between different positions, more specifically orientations, to vary the angle at which the reflected light is directed to a second optical element 14. Figure 1 shows the mirror in a first orientation (I), in a second orientation (II) and a third orientation (III). In the embodiment shown in figures 1 and 2 the second optical element 14 is a lens 14, for instance an achromatic lens. In this configuration the scanning focal point of figure 1 is created by the illumination path consisting of focusing a collimating beam in such a way that the focal point of the second optical element intersects the focal plane of the hemisphere. More specifically, lens 14 refracts the incoming collimated light beam and the resulting converging light beam is focussed in a focal point 30. The light reflected off the mirror 13 and refracted by the lens 14 is then refracted into a parallel beam 35 by the hemisphere 4. The parallel beam 35 strikes the surface 40 (figure 2) at an angle of incident (oil) large enough (more than 40 degrees (gas phase) or even more than 60 degrees (liguid phase) to cause total reflection off the surface 40. The reflected parallel light beam 36 is refracted at the water/air-hemisphere interface and focussed into a focal point 50. A third optical element 21 of the detector assembly 20 converges the incoming light into a collimated beam 53 again. The collimated beam 53 is projected onto the sensing surface of a sensing element comprising a light sensitive array, such as a CCD/CMOS camera 22.

Before the SPR scanning is started the optical elements are arranged so that expected SPR dips are within a predefined scan range (angle offsetting phase) . The optical elements need to be kept in Scheimpflug during the angle offsetting phase, for instance by using the scissor construction as described herein. Then the SPR scanning device 1 is calibrated. The pivotable mirror 13 is first tilted away from lens 14 and surface 40, such that light from light source 10 does not strike the surface 40. With camera 22 the amount of remnant light in the SPR scanning device 1 is determined and the "null"-level of the output of camera 22 is set.

In a second step the mirror 13 is tilted such that light from the light source 10 is reflected of the mirror 13 through lens 14 and the hemisphere 4 to strike the surface 40 of the sensor element 39. As mentioned earlier, the angle of incidence (oil) of the light 35 striking the surface 40 is such that total reflection (TIR) occurs at the surface 40. By means of lens 21 the light reflected off the surface 40 is imaged at the surface of camera 22. A physical property, for instance the intensity, of the light reaching the camera 22 can be determined from the output of camera 22.

At a specific angle of incidence of the incident light 35 an interaction occurs between the incident photons and the free electrons in the thin conductive layer 6 forming the sensor element 39. This interaction results in the creation of an evanescent wave in the layer of gold 114. In other words, incident photons are absorbed creating plasmons in the thin layer 6. The absorption of photons results in a reduced amount of light reflecting off the surface 40, which reduction is detected by the sensor 22.

Any material present in the band of approximately half the wavelength of the applied light above the thin layer 6 (for instance, 300 nm in this particular embodiment), for example specimens (8), may influence the refractive index at that side of the thin layer 6. If, for example, the binding of antigens (6) to antibodies (8) is to be monitored, antibodies (8) are applied to the thin layer 6. Antigens (6) are exposed to the antibodies (8) and will bind to the antibodies (8) over time. This process influences the refractive index at this side of the thin conductive layer 6. Changes in refractive index cause the angle at which maximum plasmon generation occurs to change. By monitoring this change in angle over time, a measurement of the binding rate is acguired. This change in angle is monitored by repeatedly measuring the amount of reflected light at different angles from a range of angles of incidence and determining the angle at which a minimum of reflected light is measured. Figure 4 shows a plurality of typical SPR curves generated during scans of a scanner operating in accordance with the present invention. The mirror 13 in the scanner 1 is adjusted repeatedly in order to realize the angles of incidence one by one. At each angle the reflected light intensity of every region of interest is measured and recorded. The horizontal axis 100 represents the angle of incidence. The scale in the example of figure 4 runs from -2000 millidegree till +2000 millidegree, but is alterable by the user. The zero at the axis corresponds to the middle of the scan range that was set for the scan using the optical assembly construction. This means the angles on the axis 100 are offset and the zero does not correspond to an angle of incidence of 0 degrees. The vertical axis 101 represents the reflected light intensity. The data points 102 in the graph consist of recorded reflected light intensities at the angle of incidence the measurements were taken. A smooth curve 103 has been plotted to fit the data points 102. In this particular example the data points 102 actually fit very well to the curve 103 and the minimum 104 can be calculated. In a similar way other data points 104 of the scan, associated with different regions of interest on the sensor surface, fit to further curves 105. In general different position on the sensor surface provide different reflection curves. Since the light beam impinging on the sensor surface is parallel to a great extent, the dips in the SPR curves 103-104 almost perfectly align.

In other examples the data points 102 might be scattered alongside the curve 103 to some extent. In these examples use can be made of curve-fitting technigues, such technigues as such being well known in the art.

Scanning can be performed continuous with instant grabbing images at certain angles or discontinuous, eg. when an optical fibre assembly blink subseguently at fixed angles, or scanner position is stopped at fixed angles before grabbing the reflectivity image .

Referring to figures 1 and 2, the light beam 35 projected onto the sensor surface 40 is a collimated light beam if the focal point 30 of the light 34 refracted by the lens 14 is caused to coincide with the plane of focus 37 of the hemisphere 4. The plane of focus 37 (cf. Figure 2) describes a virtual hemisphere relative to the central midpoint of the hemisphere 4, the virtual hemisphere having a radius equal to the focal distance associated with this optical element. Furthermore, since use is made of P-polarised light, only the intersection of the virtual hemispheric focal plane 37 and a polarization plane perpendicular to the hemisphere is relevant. This intersection provides a focus line (i.e. a p-focus circle) extending on the virtual plane of focus 37 of the hemisphere, on which the light from the second optical element (i.e. the lens 14) is to focus to ensure that the light striking the sensor surface 40 is fully collimated.

As is indicated in more detail in figure IB, the cone-shaped light beam 34 of the light originating from the focal point 30 in its original position (shown in continuous lines) is refracted into a cylinder-shaped light beam 35. The beam 35 is a beam of parallel light. At different angles of incidence the cone-shaped light beam is caused to originate from a different focal point, for instance a focal point 30' moved upward along the focal plane of the hemisphere 4 (shown in dotted lines) or a focal point 30'' moved downward along the same focal plane. The movement of the focal point causes a similar movement of the image of the cone light beam on the surface of the hemisphere 4 (denoted in figure IB by a small double arrow) . When during scanning of the sensor surface, i.e. during illumination of the sensor with different angles of incidence, the focal point 30 is shifted in this manner, the illuminated part or area of the sensor surface 40 remains essentially stationary. If during scanning the focal point 50 of the detector assembly is moved in a corresponding manner, the illuminated part or area of the camera 22 remains essentially stationary as well .

Referring to figure IB, the cylindrical light beam 35 "pivots" as it were around an imaginary pivot axis 38 at the sensor surface. This may result in a reduction or even prevention of the movement of the image on the sensor surface during the scanning operation .

The above movement of the focal point 30 may create complete blackening of the image (SPR condition over the whole surface area, as can be observed from figure 4 showing multiple SPR curves obtained from various regions of interest. The SPR dip positions are almost egual) .

Similarly, at the detector assembly 20 side, the light beam 36 reflected by the sensor element 39 and refracted by the hemisphere 4 (i.e. light beam 46 leaving the hemisphere 4) is focussed at a focal point 50. The reflected parallel cylindrical beam will refract to a cone into the focal point. In order to ensure that the light reaching the camera 22 is fully collimated, the focal point 50 is to be exactly aligned with the virtual hemispherical focus plane 37 of the hemisphere 4 (or, more precisely, with the above-identified p-focus-circle ) .

Accordingly, if the spherical lens 21 of the detector assembly 20 is arranged relative to the hemisphere 4 such that the focal point 50 is exactly at the focal point of the lens 21, then the light beam 46 is refracted in such a way by the lens 21 that the resulting light beam 47 received by the camera 22 is a parallel light beam.

If, for instance due to the tilting of the mirror 13 during the scanning operation from tilt position I to tilt position II (figure 2) the point of focus 30 moves away from the hemispherical plane of focus 37 of the hemisphere 4, a corresponding move of the focal point 50 at the detector side of the apparatus will occur. The result may be that the beam 47 reaching the camera 22 is not exactly collimated (parallel) anymore and that the image of the sensor surface 40 projected onto the camera 22 is somewhat shifted relative to the earlier image (i.e. the image acquired when the mirror was in its original tilt position I) . Therefore the locations of the spots 6 imaged on the camera 22 in the new situation (tilt position II) do not exactly correspond to the original position (tilt position I).

In embodiments of the invention the position (i.e the location and/or orientation) of the illumination side optics (i.e. the lens 14 and the mirror 14) is changed so that de focal point 30 is caused to remain somewhere at the plane of focus 37 of the hemisphere 4. In an embodiment the position change is only performed in the angle offsetting phase. When in the actual scanning phase the optics remain stationary. In other embodiments the position change may be performed both in the angle offsetting phase and the scanning phase.

According to an embodiment of the present invention, means are provided to effect the above mentioned position change. For instance, the lens 14 may be moved mechanically to a new position wherein it is able to focus the light beam 34 on the plane of focus 37. For instance, the lens 14 may be mounted to a scissor construction (cf . figure 11) .

Similarly, at the side of the detector assembly 20, the light beam 46 leaving the hemisphere 4 is focussed at a focal point 50 which should be exactly aligned with the hemispherical focus plane 37 of the hemisphere 4. When the lens 21 is arranged at the focal distance relative to the hemisphere 4 such that the focal point of the lens 21 exactly coincides with the focal point 50, then the light beam 46 may be refracted in such a way by the lens 21 that the resulting light beam 47 is perfectly collimated.

However, if, for instance, due to the tilting of the mirror 13, the point of focus 50 has first moved away from the hemispherical plane of focus 37 and has subsequently been moved back onto the plane of focus 37 by a movement of the illumination side optics, the position of focal point 50 has been shifted along the plane of focus 37. The position of the focal point may be moved and set by changing the optical configuration in the angle offsetting phase. The result of this shift may be that the light beam 47 reaching the camera 22 is not exactly collimated (parallel) anymore. The effect may be that the image of the sensor surface 40 projected onto the camera 22 is somewhat shifted relative to the earlier image (i.e. the image acguired when the mirror was in its original orientation I) . Therefore the position of the spots 8 imaged on the camera 22 (spots 19 in figure 3) in the new situation (II) would not exactly correspond to the original position (in situation I).

In order to keep a perfectly collimated beam of light, in accordance with embodiments of the invention, the positions of the one or more elements of the detector assembly 20 should be altered so that the focal point of the spherical lens 21 and the focal point of the hemisphere 4 coincide again. This could be accomplished by mechanical means (not shown in the drawings.

To further improve the guality of the image on the camera 22, the optical elements at the detector assembly side should be arranged (oriented) to comply with the so-called Scheimpflug principle. In the situation shown in figure 2, the spherical lens 21, camera 22 and sensor element 39 are in the Scheimpflug condition (i.e. in a condition wherein the

Scheimpflug principle is complied with) . In other words, the imaginary tangent line 60 extended from the lens plane 31 of the lens 21, the imaginary tangent line 62 of the camera plane 63 of the camera 22 and the imaginary tangent line 68 extended from the sensor element 39 (as is shown in figure 2) intersects in one common point, the Scheimpflug intersection point 65. In this condition the image of the planar surface 40 of the sensor element 39 on the camera 22 is in focus. A similar configuration can be accomplished at the illumination side of the apparatus. Figure 2 shows that the imaginary tangent line 68 of the lens plane 69 of the lens 14, the imaginary tangent line of the mirror 13 (in the tilt position I) and the imaginary tangent line 68 extended from the sensor element 39, intersect in one common point of intersection or Scheimpflug intersection point 66. In this condition the image of the mirror surface of the mirror 13 on the sensor surface 40 can be made completely in focus .

When the mirror 13 is tilted from the tilt position I to tilt position II, the Scheimpflug intersection point 66 is shifted in direction R (figure 2) to a new Scheimpflug point 66' . Therefore, in order to keep the illumination assembly in Scheimpflug condition, the lens 14 should be tilted somewhat, as is indicated with dotted line 68'.

Similarly at the detector assembly side, the

Scheimpflug point 65 is shifted (R2) to a new Scheimpflug point 65'. This means that the camera 22 and the spherical lens 21 should be rotated in order to bring the intersection point of lines 60' and 62' into correspondence with the new Scheimpflug point 65', as is shown in figure 5. The situation wherein the Scheimpflug condition is restored is shown in figure 5.

Generally the lens 21 is positioned closer to the line 68 than the camera 22. Accordingly, the rotation angle βι between lines 60 and 60' generally is smaller than the rotation angle β2 between lines 62 and 62' (β2 = 0,5 times βι, for instance) . This means that the camera needs to be rotated less than the spherical lens, at least during the actual mirror scanning process . In other situations, for instance during the angle offsetting procedure, the camera may need to be rotated more than the spherical lens . The rotation of the camera and spherical lens during the angle offsetting procedure is described more detailed in connection with figure 11. In other embodiments of the invention only one of the camera/mirror and the spherical lens is rotated. Other embodiments only tilt the camera position during angle off-set and not during mirror scanning. All other embodiments of combinations of with or without tilting the illumination path and /or detection path can be considered and can be assumed according to the invention. Although the Scheimpflug condition is not reached to its full extent in these embodiments, the guality of the image on the camera 20 may improve considerably.

In further embodiments of the present invention the

Scheimpflug orientation of the camera and/or the lens 21 is accomplished by a mechanical construction. Referring to figure 10 the mechanical construction may comprise a support element 150 that may be forced to move along a guide 151 (for instance a rail) extending along the earlier mentioned tangent line 68 of the sensor element 39. A first support 152 arm may be connected to camera 22. The spherical lens 21 may be connected to the same support arm 152. In the embodiment of figure 10 a separate support art 153 is provided to connect the spherical lens 21 to the support element 150. If the support arms are both hingedly attached to the support element at one common point, the optical elements 22, 21 can easily be held in a correct Scheimpflug condition by simply moving the support element along the guide. Using this mechanical construction along a guide a change in the off-set position will result also in an automatic change of camera and lens in a correct tilted position according to the Scheimpflug condition.

In further embodiments the illumination assembly and/or the detection assembly comprises an element provided with at least one aperture which is a hole or an opening through which light travels. More specifically, the aperture is the opening or pinhole that determines the cone angle of the bundle of light rays that come to a focus in the image plane. The aperture determines how collimated the rays admitted to the first optical hemispherical element are. This has a positive influence on the appearance at the image plane. By changing the aperture position in the focal plane, a cone of light is generated. The light travels along the optical axis to the optical element and once arrived inside the optical element remains highly parallel . Additionally a parallel (cylindrical) beam may be reflected from the sensor surface. This may create a reflected parallel cylindrical beam which is transformed to a cone of light which travels to the second focal point. From the second focal point a reversed cone of light travels to the lens. Scanning of the coned light will occur over the lens and an image is formed at a distinct position from this lens. The changed distance of travelled light from each point of the surface of the sensor surface forces the sensing element (camera) and lens both in a suitable relative angle (in accordance with the Scheimpflug condition) . The tilted sensing element and lens position may egualize the distance to get for each point on the sensor surface a sharp image on the sensing element (camera) .

Figure 6 illustrates a further embodiment of the present invention. The diverging light beam for illumination of the hemisphere 4 is generated in this embodiment by an optical fibre 115. The fibre acts as a waveguide to transmit light between the two ends of the fiber. At the proximal end of the fibre 115 a light source 10 is connected. At the distal end 116 of the optical fibre a diverging light beam 117 is generated. The diverging light beam is directed towards the hemisphere 4. In this configuration the illumination assembly focal point 118 is situated at or close to the end 116 of the optical fibre. During scanning the focal point 118 may be displaced (P6 - P7) by a displacement unit (not explicitly shown, for instance a frame driven by one or more electric motors or a similar construction) to coincide with the above-references P-focus circle in order to maintain a parallel beam inside the hemisphere 4.

In another embodiment shown in figure 7 the second optical element is a non-transparent plate 120 provided with an aperture

121. The plate 120 may be illuminated by a remote light source

122, for instance one or more LED lights. The light source in combination with the plate generates a diverging light beam 123 having a focal point 124 at or close to the aperture 121. By moving the aperture of the place the position of the illumination assembly focal point 124 move be suitable placed on the p-focus circle 125.

Referring to figure 8 an embodiment in shown wherein the second optical element comprises a plate 130 shaped so as to coincide with the focus plane 37 (more specifically, the p-focus line 125) of the hemisphere 4. The plate 130 is provided with a plurality of apertures 131-133, arranged so as to coincide with the p-focus circle 125. Behind the apertures 131-133 a number of light elements, for instance LED's 134-136, are arranged that can be selectively switched on and off by a controller 137. By controlling the light elements the apertures may be lighted consecutively so that a diverging light beam 137 may be generated from different positions (and corresponding different angles of incidence on the sensor surface) relative to the hemisphere. Accordingly, the light beam 137 may be used to scan the sensor surface of the hemisphere 4 at different angles of incidence without any actual movement of the optical element.

Referring to figure 9 a further embodiment is shown. In this embodiment use is made of a plurality of light fibres 140, 141, 142. The distal ends of the fibres arranged at the p-focus circle 125. A controller 139 is connected to fibres 140-142 which acts as a light source to selectively generate a light beam 143 from one of the fibres 140-142. In this embodiment displacement of the illumination assembly focal point 144 can be accomplished easily by switching the fibres 140-142 consecutively on and off.

In the embodiments described above the optical elements are arranged to correspond to the Scheimpflug condition during the scanning operation (i.e. during the angle offsetting phase and/or actual scanning phase) . The Scheimpflug condition should also be maintained when the entire optical constructions has been displaced, for instance when the apparatus is rotated in order to enable different kind of measurements to take place. For instance, in a gas measurement set-up the measurements are to be performed at an angle of about 40 degrees, while in the liguid measurement set-up the measurements should be performed at an angle of about (at least) 65 degrees. Furthermore, if different types of prism material are used, for instance SF10 instead of BK7, other starting angles should be set.

Figure 11 shows another embodiment of the present invention. In this embodiment the apparatus comprises a frame having a tiltable pair of scissors/frame elements at which the illumination element and sensing element and/or the first and third optical element are attached. The optical components 4, 13, 14, 21, 22 are mounted on a generally rhomboidal pivotable arm construction or scissor construction 155.

Construction 155 comprises two pivotable arms 160, 161 for attaching the tiltable mirror 13 and the camera 22, respectively. Arms 160, 162 may be rotated along a pivot 170. In order to force both arms 160,162 to pivot simultaneously, further arms 164,166 are provided. At one end arms 164,166 are pivotably mounted to arms 160, 162 respectively, while at the opposite end arms 164, 166 are pivotable attached to one another .

Assuming the position of the hemisphere 4 is stationary, then rotation (Rn) of a spindle 156 in a coarse tuning phase results in the upward (Pu) or downward (PD) movement of the pivot 158 and therefore in a corresponding movement of the optical arms 160,162. If the length of the arms 160,162 are suitably chosen, the operation of the spindle may enable the optical elements and the sensing element to be arranged relatively to each other in such a way that during the subseguent scanning operation the focal points of the illumination assembly and the detector assembly may more easily be moved substantially along the focal plane of the hemisphere 4.

More specifically, rotation of the spindle 156 results in a translating movement of the tiltable mirror 13 and the sensing element 22 and hence in a change of the location of the mirror and the sensing element relative to the first optical hemispherical element 170. Rotation of the spindle 156 also results in change of the location of the second optical element 14 and the third optical element 21 respectively attached to frame elements (arms) 160 and 162. However, the rotation of the spindle additionally causes the orientation of the second and third optical elements with respect to the first optical hemispherical element to change.

If the optical elements were to be attached fixedly to the arms 160,162, then the operation of the spindle 156 might cause the elements to lose their optimal Scheimpflug condition. In order to restore the Scheimpflug condition, use can therefore be made of the detector side orientation adjustment unit 160 of figure 10 (and a corresponding unit 161 for the illumination side of the system) .

Prior to the angle off-setting phase and the actual scanning phase the optical elements and the sensing elements are tuned to provide the improved scanning results in the scanning phase. The above-described rotation of the spindle enables a first, coarse tuning of the optical system. In embodiments of the present invention the scanning results to be obtained by the apparatus may be further improved by fine tuning the optical system, for instance by changing the orientation of the sensing element and one or more of the second and third optical elements.

Accordingly, in embodiments of the invention, the detector assembly comprises an orientation adjustment unit 150 for adjusting the orientation of the sensing element (or at least the sensing area thereof), the third optical element or both the sensing element and the third optical element in a fine tuning phase so as to further tune the optical system.

Additionally or alternatively, in embodiments of the invention, the illumination assembly comprises an orientation adjustment unit 161 for adjusting the orientation of the second optical element so as to further tune the optical system.

Any of the orientation adjustment units may be embodied so that the orientation of the elements is performed manually, for instance by the operator of the apparatus. In further embodiments of the present invention at least one of the adjustment units may be driven by one or more electric motors controlled by a control module. The control module is also connected to a sensor, for instance the sensing element 22 and/or a separate optical sensor. The optical signal detected by the sensor is converted into an electrical signal and sent to the control module. Based on the electrical signal received from the sensor the control module may control the electric motors to adjust the orientation of the sensing element and/or the second optical element and/or the third optical element.

The alignment can be performed prior to the angle offsetting phase, during the angle offsetting phase and/or in the actual scanning phase. Especially in the angle offsetting procedure (involving relatively large angles) the arm construction 155 may enable the illumination assembly and detector assembly to be positioned easily while keeping the optical elements at the right distance from the hemisphere.

In embodiments of the invention the translational and tilting movement of the elements of the detector assembly is performed simultaneously with the movement of the element (s) of the illumination assembly, for instance in the embodiment shown in figure 11. In other embodiments the elements of the illumination assembly may be moved essentially independently from the elements of the detection assembly. This makes it for instance possible to move the sensing element and third optical element independently in Scheimpflug condition at both optical paths .

The present invention is not limited to the specific embodiments described herein; the rights sought are defined by the following claims, within the scope of which many

modifications can be envisaged.

Claims

1. Apparatus for surface plasmon resonance (SPR) scanning, the apparatus comprising:
- a sensor assembly comprising:
- an essentially hemispherical first optical element, substantially transparent to light and defining a first optical element focal plane and a sensor surface;
- an SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical hemispherical element;
- an illumination assembly arranged to direct light diverging from an illumination assembly focal point to the sensor surface of the first optical element, the light arriving at the sensor surface at varying angles of incidence as a substantially collimated light beam;
- a detector assembly for the spatially resolved acguisition of a light property, for instance the intensity, of light reflected at the sensor surface of the first optical element at varying angles of incidence, the detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element having a third optical element focal plane;
wherein the detector assembly is configured to adapt the location and orientation of at least one of the sensing element and third optical element, preferably both the sensing element and the third optical element, to focus the reflected light in a focal point located at both the first focal plane of the first optical hemispherical element of the sensor assembly and the third focal plane of the third optical element of the detector assembly.
2. Apparatus as claimed in claim 1, wherein the detector assembly comprises an orientation adjustment unit for adjusting the orientation of the sensing element and/or the third optical element with respect to the first optical hemispherical element.
3. Apparatus as claimed in any of the preceding claims, wherein the first optical element and the third optical element, the first optical element and the sensing element, and/or the first optical element, the third optical element and the sensing element are essentially in Scheimpflug condition.
4. Apparatus as claimed in any of the preceding claims, wherein the sensing element and third optical element are mounted so as to be individually tiltable .
5. Apparatus for surface plasmon resonance (SPR) scanning, preferably an apparatus as claimed in any of the preceding claims, the apparatus comprising:
- a sensor assembly comprising:
- an essentially hemispherical first optical element, substantially transparent to light and defining a first optical element focal plane and a sensor surface;
- an SPR sensor element comprising a thin layer of conductive material, at least partially provided at the sensor surface of the first optical element;
- an illumination assembly arranged to direct light diverging from an illumination assembly focal point to the sensor surface of the first optical hemispherical element, the light arriving at the sensor surface at varying angles of incidence as a substantially collimated light beam;
- a detector assembly for the spatially resolved acguisition of a light property, for instance the intensity, of light reflected at the sensor surface of the first optical hemispherical element at varying angles of incidence, the detector assembly comprising a sensing element for sensing the image of the illuminated sensor surface and a third optical element having a third optical element focal plane;
wherein the illumination assembly is configured to move the position of the illumination assembly focal point during scanning of the sensor surface at varying angles of incidence so as to essentially align this position with the first optical hemispherical element focal plane.
6. Apparatus as claimed in claim 5, wherein the illumination assembly is configured to move the illumination assembly focal point along the first optical hemispherical element focal plane in a manner so as to reduce or even prevent movement of the illuminated area of the sensor surface.
7. Apparatus as claimed in claim 5 or 6, wherein the detector assembly is configured to change the position, preferably both the location and the orientation, of the third optical element and/or the sensing element so as to at least partly compensate for the movement of the image on the sensing element as a result of movement of the position of the illumination assembly focal point, wherein the position of the third optical element and/or the sensing element preferably is changed so as to minimize the movement of the image on the sensing element.
8. Apparatus as claimed in any of the preceding claims, wherein the illumination assembly comprises a tiltable mirror for controlling the angle of incidence and a second optical element to focus light from the tiltable mirror in the illumination assembly focal point.
9. Apparatus as claimed in any of the preceding claims, wherein the illumination assembly comprises an orientation adjustment unit for adjusting the orientation of the second optical element with respect to the first optical hemispherical element .
10. Apparatus as claimed in claim 8 or 9, wherein the second optical element and the first optical element, the tiltable mirror and the first optical hemispherical element, and/or the fist optical element, the second optical element and the tiltable mirror are essentially maintained in Scheimpflug condition.
11. Apparatus as claimed in claim 10, wherein Scheimpflug condition is maintained during a sensor surface scan.
12. Apparatus as claimed in any of the preceding claims, wherein at least a sensing area of the sensing element is moved essentially in synchronisation with the movement of the tiltable mirror .
13. Apparatus as claimed in any of the preceding claims, wherein the detector assembly is arranged so that an imaginary axis extended from the sensor surface, an imaginary axis extended from the third optical element, orthogonal to its optical axis, and an imaginary axis extended from the sensing element, orthogonal to the optical axis of the sensing element, intersect in essentially one common point.
14. Apparatus as claimed in any of the preceding claims, comprising :
- a support element arranged so as to be movable along a guide extending along an imaginary axis extended from the sensor surface, - a first support arm connected to the sensing element,
- a second support arm connected to the third optical element ;
wherein the first and second support arms are pivotably connected to the support element.
15. Apparatus as claimed in any of the preceding claims, comprising :
- a support element arranged so as to be movable along a guide extending along an imaginary axis extended from the sensor surface,
- a first support arm connected to the mirror,
- a second support arm connected to the second optical element ;
wherein the first and second support arms are pivotably connected to the support element.
16. Apparatus as claimed in any of the preceding claims, comprising a frame having a tiltable pair of scissors frame elements at which the illumination and sensing element, preferably also the first and third optical element, are attached .
17. Apparatus as claimed in any of the preceding claims, wherein the support arms are pivotable with respect to a common pivot point .
18. Apparatus as claimed in any of the preceding claims, comprising a drive construction for driving the support element along the guide and a controller for controlling the drive construction to arrange the support element at the Scheimpflug point .
19. Apparatus as claimed in any of the preceding claims, wherein the illumination assembly comprises an angle controller for controlling the angle of incidence of the light beam striking the sensor surface.
20. Apparatus as claimed in any of the preceding claims, wherein the second optical element is an essentially
non-transparent component provided with a plurality of pinholes, which pinholes may be selectively made transparent or non-transparent to displace the position of the second element focal point.
21. Apparatus as claimed in any of the preceding claims, wherein the light source and second optical element are combined to form essentially a component comprising a plurality of optical fibres, which optical fibres may be selectively and subseguently switched on and off to displace the position of the second element focal point.
22. Apparatus as claimed in any of the preceding claims, wherein the second optical element is a pinhole transparent for light or an optical fibre and wherein the second optical element is arranged to be displaced along an optical plane to displace the position of the second element focal point.
23. Apparatus as claimed in claim 22, wherein the pinholes and/or optical fibres are situated at a virtual hemispherical focus plane relative to the optical centre of the first optical hemispherical element.
24. Apparatus as claimed in any of the preceding claims, wherein the SPR sensor element comprises an array of regions of interest (ROI's) to be scanned at a range of angles of incidence and to be imaged on the sensing element.
25. Apparatus as claimed in any of the preceding claims, comprising a fluidic system for delivering and exposing a sample medium to at least the sensor element.
26. Apparatus as claimed in any of the preceding claims, wherein the sensor element is spotted with one or more different ligands, the spots forming regions of interest to be scanned.
27. Apparatus as claimed in any of the preceding claims, wherein the light source is a light emitting diode (LED) mounted on an optical fibre acting as a pinhole.
28. Apparatus as claimed in any of the preceding claims, wherein the sensor element is removably attached to the first optical hemispherical element.
29. Apparatus as claimed in any of the preceding claims, wherein the first optical hemispherical element is optically matched to a disposable sensor element, preferably using an optogel or refractive index matching oil.
30. Apparatus as claimed in any of the preceding claims, wherein the first optical hemispherical element is a disposable.
31. Apparatus as claimed in any of the previous claims, wherein the light source comprises a mirror reflecting light from an array of light emitting diodes (LED's).
32. Apparatus as claimed in any of the previous claims, wherein in use the first optical hemispherical element is horizontally oriented and the sensor element is arranged at the top side of the first optical hemispherical element.
33. Apparatus as claimed in any of the previous claims, wherein the second optical element and/or the third optical element comprise an aspherical lens.
34. Apparatus as claimed in any of the preceding claims, comprising a control module configured to control the orientation, preferably also the location, of at least one of the second optical element, the third optical element and the sensing element based on a signal received from the sensing element.
35. Apparatus as claimed in any of the preceding claims, wherein at least one of the orientation adjustment units comprises a drive motor connected to a control module, the control module being connected to the sensing element and being configured to tune the orientation of the sensing element and/or at least one of the optical elements based on a signal received from the sensing element.
36. Method for surface plasmon resonance (SPR) scanning, the method comprising:
- angle scanning a surface of an SPR sensor element provided at the sensor surface of an essentially hemispherical first optical element by illuminating the sensor surface at varying angles of incidence with a substantially collimated light beam
- measuring a light property, for instance the intensity, of light reflected at the sensor surface by sensing the image of the illuminated sensor surface, received from a third optical element, by a sensing element;
the method further comprising:
- adapting the position of at least one of the sensing element and third element during scanning so as to focus the reflected light in a focal point located at both the first focus plane of the first element of the sensor assembly and the third focal plane of the third element of the detector assembly.
37. Method for surface plasmon resonance (SPR) scanning, preferably a method as claimed in claim 36, the method comprising:
- angle scanning a surface of an SPR sensor element provided at the sensor surface of an essentially hemispherical first optical element by illuminating the sensor surface at varying angles of incidence with a substantially collimated light beam
- measuring a light property, for instance the intensity, of light reflected at the sensor surface by sensing the image of the illuminated sensor surface, received from a third optical element, by a sensing element;
the method further comprising:
- adapting the position of the illumination assembly focal point during scanning of the sensor surface at varying angles of incidence so as to essentially align this position with the first optical hemispherical element focal plane.
38. Method as claimed in claim 36 and/or 37, wherein an apparatus as claimed in any of the preceding claims 1-35 is used.
39. Use of an apparatus as claimed in any of claims 1-35.
PCT/NL2011/050806 2010-11-25 2011-11-25 Method and apparatus for surface plasmon resonance angle scanning WO2012070942A1 (en)

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