GB2516277A

GB2516277A - Optical apparatus and methods

Info

Publication number: GB2516277A
Application number: GB1312795.6A
Authority: GB
Inventors: Robert Jones; Alfred Newman; Martin Brock; Dean Stuart Griffiths
Original assignee: Cambridge Consultants Ltd
Current assignee: Cambridge Consultants Ltd
Priority date: 2013-07-17
Filing date: 2013-07-17
Publication date: 2015-01-21
Also published as: GB201312795D0

Abstract

Optical apparatus for measuring characteristics of a measurement object comprising an illumination portion, detection portion and processing portion. The illumination portion produces at least one pair of spatially separated areas of illumination for illuminating a measurement object to produce an associated light field. The light field produced by illumination of the surface of the measurement object comprises a component corresponding to interference between the areas of illumination. illuminates a measurement site on the measurement object and illuminates a reference site on the measurement object. The detection portion receives light from the measurement object, directs the received light onto a detector, outputs signals from the detector dependent on the intensity of the detected light. The processing portion analyses the signals output by the detecting means to measure the characteristics of the measurement object.

Description

I

Optical Apparatus and Methods The present invention relates to optical apparatus and associated methods. The invention has particular although not exclusive relevance to an interferometer for measuring any of a plurality of parameters (e.g. vibration amplitude/frequency, refractive index, surface profile etc.) of a measurement object in harsh environments in which there are typically a number of confounding factors.

Speckle pattern interferoriietry (SPI) uses interference characteristics of electromagnetic waves incident on a measurement object to measure the characteristics of that measurement object. In conventional techniques, an SPI sensor will typically illuminate a measurement object with a sample beam comprising laser light. The measurement object must have an optically rough surface so that when it is illuminated by the laser light an image comprising an associated speckle pattern is formed. A reference' beam is derived from the same laser beam as the sample beam and is superimposed on the image from the measurement object. The light from the measurement object and the light of the reference beam interfere to produce a corresponding interference speckle pattern, which changes with out-of-plane displacement of the measurement object as a result of changes in the phase of the light from the measurement object relative to that of the reference beam. The changes in the speckle pattern can therefore be monitored, recorded and analysed to measure static and dynamic displacements of the measurement object.

The speckle pattern produced and analysed in such systems is a subjective speckle pattern which varies in dependence on viewing parameters such as, for example, lens aperture, position and/or the like.

Sheared beam interferometry (or sheared interferometry) is a technique in which a light wavefront is split (or sheared') into two images which overlap to cause interference with one another to provide a plurality of fringes which may be used to determine the characteristics of a measurement object. One example of sheared beam interferometry has been described previously for applications in speckle pattern interferometry (SPI), for example R Jones and C Wykes: Holographic and Speckle Interferometry, Cambridge Series in Modern Optics 6, CUP 1983, pp. 156 - 159. In this example light incident on a surface produces a speckle pattern image which is split, by a shearing interferometer, into two interfering images to produce an interference pattern that may be observed through the interferometer.

A specific configuration of common path shearing Interferometry based on an angled wedge illumination element is the subject of an earlier patent application by Cambridge Consultants (WO 03/012366A1, published 13 February 2003).

More recently, double lateral shearing interferometry has been used for ophthalmic measurements of tear film topography: Alfred Dubra et al, 1 March 2004/ vo148, No 7/Applied Optics: pp. 1191-1199.

Measurement of the rotation of optically rough objects using purely laser speckle (without a generated fringe field, or spatially controllable differential measurement) is the subject of a patent by Zeev Zalevsky (WO 09/013738).

However, the above techniques have a number of limitations which make it difficult, or impossible, for them to be used to measure precisely a full range of parameters associated with a measurement object (such as vibration amplitude/frequency, refractive index, surface profile etc.), with high phase resolution (i.e. typically of the order io radians), in the presence of common confounding factors including, for example, high levels of background vibration, temperature and atmospheric turbulence, and higher order surface motions. Any such confounding factor would normally prevent the operation of conventional interferometers and therefore make them unsuitable for many measurement environments.

Accordingly, preferred embodiments of the present invention aim to provide methods and apparatus which overcome or at least alleviate one or more of the above issues.

In one aspect of the invention there is provided optical apparatus for measuring characteristics of a measurement object, the apparatus comprising an illumination portion and, detection portion and a processing portion: the illumination portion comprising: means for producing at least one pair of spatially separated areas of illumination for illuminating a surface of the measurement object to produce an associated light field from which the characteristics of the measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: when the surface of the measurement object is optically rough, a component associated with self-interference within at least one of the areas of illumination; and a component corresponding to interference between the areas of illumination which is separable from any component comprising interference associated with self-interference; the detection portion comprising: means for detecting light and for outputting signals dependent on the intensity of the detected light; means for receiving the light field from the measurement object resulting from the illumination of the measurement object with the at least one pair of the spatially separated areas of illumination, the light field resulting from each pair containing at least the component corresponding to interference between the areas of illumination; means for directing the received light field onto the light detecting means; the processing portion comprising: means for analysing the signals output by the detecting means to measure the characteristics of the measurement object.

The means for producing the at least one pair of spatially separated areas of illumination may comprise shearing optics for shearing an incoming beam of light into at least two sheared beams of mutually coherent light, each sheared beam representing a respective source of one of the spatially separated areas of illumination.

The optical apparatus may further comprise optics for transforming the at least two sheared beams into at least two parallel beams each parallel beam representing a respective source of one of the spatially separated areas of illumination.

The light field may comprise a plurality components (e.g. in the form of diffraction fringes) having an increased power at spatial frequencies corresponding to the interference between the areas of illumination.

The analysing means may be operable to analyse the signals output by the detecting means to determine changes in the components having an increased power and/or to measure a difference between a first phase of one of the at least one of the areas of illumination and a second phase for the other of the areas of illumination based on, for example, the determined changes in the components having an increased power.

The analysing means may be operable to analyse the signals output by the detecting means, for example to measure characteristics of the surface of the measurement object associated with an effective difference between an optical path length for at least one of the areas of illumination and an optical path length for another of the areas of illumination.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics comprising a rotation of the surface of the measurement object to cause the effective difference between an optical path length for at least one of the areas of illumination and an optical path length for the other of the areas of illumination.

The illuminated surface of the measurement object may be an optically rough surface, the light field from the measurement object may comprise at least one component comprising self-interference associated with roughness of the optically rough surface (e.g. a speckle pattern), and the analysing means may be operable to discriminate between the component corresponding to interference between the areas of illumination and the component comprising self-interference associated with roughness of the optically rough surface, whereby to measure the characteristics of the measurement object.

The analysing means may be operable to analyse the self-interference associated with roughness of the optically rough surface for example to measure the characteristics of the measurement object.

The analysing means may be operable to analyse the self-interference associated with roughness of the optically rough surface to measure characteristics of the measurement object associated with a movement of the illuminated surface of the measurement object (e.g. a translational movement in the plane of the illumination).

The analysing means may be operable to analyse the self-interference associated with roughness of the optically rough surface to measure characteristics of the measurement object associated with a movement, of the illuminated surface of the measurement object with components in either or both of two axial directions within the plane of the measurement surface.

The analysing means may be operable to analyse the self-interference associated with roughness of the optically rough surface to measure characteristics of the measurement object associated with a rotational movement, of the illuminated surface of the measurement object, about an axis normal to the plane of the measurement surface based on measurements of differential translations at two separate locations.

The means for producing spatially separated areas of illumination may be operable to illuminate a measurement object with at least three spatially separated areas of illumination, wherein the at least three spatially separated areas of illumination are arranged to allow measurement for the measurement object to be performed for each of at least two axis of rotation.

The detection portion may comprise means for spatially filtering the light field associated with the at least three spatially separated areas of illumination to produce a light field associated with two of the spatially separated areas of illumination whereby to select an axis of rotation for which measurement is to be performed.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics comprising a rotation of a surface of the measurement object about the selected axis.

The detecting means may comprise a point detector.

The optical apparatus may further comprise means for modulating phase of at least one of the spatially separated areas of illumination, using a known phase modulation, whereby to allow the analysing means to determine differences in phase associated with characteristics of the measurement object by analysing phased with reference to the known phase modulation.

The detecting means may comprise a one dimensional detector (e.g. a linear detector or linear array detector).

The detecting means may comprise a two dimensional detector.

The means for producing at least one pair of spatially separated areas of illumination may be operable to provide the spatially separated areas of illumination as two spots of illumination on a surface of a measurement object.

The means for producing at least one pair of spatially separated areas of illumination may be operable to provide the spatially separated areas of illumination as two lines of illumination.

The analysing means may be operable to analyse respective signals output by the detecting means for each of a plurality of different parts of the lines of illumination, whereby to measure characteristics of the measurement object at a plurality of different locations, each location being associated with a different respective part of the lines of illumination.

The means for producing at least one pair of spatially separated areas of illumination may comprise means for scanning the spatially separated areas of illumination across a measurement object (e.g. without moving the apparatus from one location to another).

The scanning means may comprise at least one mirror.

The scanning means may comprise at least one scanning lens (e.g. an F over theta lens).

The means for producing at least one pair of spatially separated areas of illumination may be operable to: illuminate a measurement site on a measurement object with at least one of the spatially separated areas of illumination; and/or illuminate a reference site on a measurement object with at least one other of the spatially separated areas of illumination.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics of the measurement object associated with an effective difference between: an optical path length for the at least one area of illumination illuminating the measurement site; and an optical path length for the at least one other area of illumination illuminating the reference site.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics, of the measurement object, associated with molecular surface binding at the measurement site.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics, of the measurement object, associated with the occurrence of binding events associated with a change in optical path length.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics, of the measurement object, associated with the occurrence of binding events associated with an increase in optical path length.

The analysing means may be operable to analyse the signals output by the detecting means to measure characteristics, of the measurement object, associated with the occurrence of binding events associated with a decrease in optical path length.

The means for producing at least one pair of spatially separated areas of illumination may be operable to illuminate at least two further reference sites on the measurement object with at least one further pair of spatially separated areas of illumination; wherein the analysing means may be operable to analyse the signals output by the detecting means for illumination incident on the at least two further reference sites to measure characteristics, of the measurement object, associated with rotation of the measurement object; and wherein the analysing means may be operable to use the measured characteristics associated with rotation of the measurement object to mitigate the effect of the rotation the measures characteristics associated with molecular surface binding.

The optical apparatus may further comprise means for inducing surface plasmon resonance while performing the measurement.

The measurement object may be located in an optically transparent medium (e.g. a medium having a refractive index greater than or equal to 1) and the illumination and detection portions may be provided on either side of the optically transparent medium.

The measurement object may be located in an optically transparent medium (e.g. a medium having a refractive index greater than or equal to 1) and the illumination and detection portions may be provided on the same side of the optically transparent medium.

The measurement object may be optically transparent having a refractive index that may be different to the refractive index of the transparent medium.

The analysing means may be operable to measure characteristics of the measurement object based on differences in phase associated with differences in the refractive indexes.

The analysing means may be operable to measure characteristics of a measurement object comprising a particle flowing in the transparent medium, past the areas of illumination, the characteristics comprising a size of the particle.

In one aspect of the invention there is provided illumination apparatus for use as the illumination portion of the optical apparatus, the illumination apparatus comprising: the means for producing at least one pair of spatially separated areas of illumination for use in measuring the characteristics of the measurement object, wherein the areas of illumination may be mutually coherent and may each be provided via a substantially common path.

In one aspect of the invention there is provided detection apparatus for use as the detection portion, of the optical apparatus, the detection apparatus comprising: the means for detecting light and for outputting a signal dependent on the intensity of the detected light; the means for receiving a light field from the measurement object resulting from illumination of the measurement object with at least one of the spatially separated areas of illumination; and/or the means for directing the received light field onto the light detecting means.ln one aspect of the invention there is provided signal processing apparatus for use as said processing portion, of the optical apparatus, the signal processing apparatus comprising said means for analysing said signals output by said detecting means to measure said characteristics of said measurement object.

In one aspect of the invention there is provided a method performed by optical apparatus for measuring characteristics of a measurement object, the apparatus comprising an illumination portion and, detection portion and a processing portion, the method comprising: the illumination portion: producing at least one pair of spatially separated areas of illumination for illuminating a surface of said measurement object to produce an associated light field from which said characteristics of said measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: when said surface of the measurement object is optically rough, a component associated with self-interference within at least one of said areas of illumination; and a component corresponding to interference between said areas of illumination which is separable from any component comprising interference associated with self-interference; the detection portion: receiving said light field from the measurement object resulting from said illumination of the measurement object with the at least one pair of said spatially separated areas of illumination, the light field resulting from each pair containing at least said component corresponding to interference between said areas of illumination; directing the received light field onto light detecting means; detecting light at the detecting means and outputting signals dependent on the intensity of the detected light; the processing portion: analysing said signals output by said detecting means to measure said characteristics of said measurement object.

In one aspect of the invention there is provided a method performed by illumination apparatus, the method comprising: producing at least one pair of spatially separated areas of illumination for illuminating a surface of a measurement object to produce an associated light field from which said characteristics of said measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: when said surface of the measurement object is optically rough, a component associated with self-interference within at least one of said areas of illumination; and a component corresponding to interference between said areas of illumination which is separable from any component comprising interference associated with self-interference.

In one aspect of the invention there is provided a method performed by detection apparatus for detecting a light field produced using the method performed by the illumination apparatus, the method performed by the detection apparatus comprising: receiving said light field from the measurement object resulting from said illumination of the measurement object with the at least one pair of said spatially separated areas of illumination, the light field resulting from each pair containing at least said component corresponding to interference between said areas of illumination.

In one aspect of the invention there is provided a method performed by signal processing apparatus for processing signals output as part of the method performed by the detection apparatus, the method performed by the signal processing apparatus comprising: analysing said signals output by said detecting means to measure said characteristics of said measurement object.

Aspects of the invention are recited in the appended independent claims.

Specific areas of application described in detail in this document are remote motion measurement, and molecular binding detection.

Each feature disclosed in this specification (which term includes the claims) and/or shown in the drawings may be incorporated in the invention independently (or in combination with) any other disclosed and/or illustrated features. In particular but without limitation the features of any of the claims dependent from a particular independent claim may be introduced into that independent claim in any combination or individually.

Embodiments of the invention will now be described by way of example only with reference to the attached figures in which: Figure 1 shows a general configuration of exemplary interferometer apparatus; Figure 2 shows beam shearing optics that are suitable for use in the interferometer apparatus of Figure 1; and Figures 3(a) and 3(b) show, in different respective planes, detection optics that are suitable for use in the interferometer apparatus of Figure 1; Figure 4 shows an exemplary representation of how, for optically rough surfaces, carrier fringe field may be superimposed on a speckle pattern using the interferometer apparatus of Figure 1; Figure 5 illustrates the potential use of the interferonieter apparatus of Figure 1 to measure rotational movement of a surface of a measurement object; Figure 6 shows an exemplary spatial power spectrum of a one dimensional sensed image provided by a linear array, in the interferometer apparatus of Figure 1, for an optically smooth surface and an optically rough surface; Figure 7 illustrates the effect of a tangential translation of a measurement object on speckle envelope and fringe patterns for that object; Figure 8 shows another example of beam shearing optics that are suitable for use in the interferometer apparatus of Figure 1; Figure 9 shows another configuration of exemplary interferometer apparatus that may be used to enable measurements to be performed sequentially over a two dimensional (2D) surface; Figure 10 shows part of the configuration shown in Figure 9 and illustrates operation of that configuration to scan a measurement surface; Figures 11(a) and (b) show, in different respective planes, a further arrangement of detection optics that are suitable for use in the interferometer apparatus of Figure 1; Figure 12 shows a four-spot interferometer apparatus which can provide measurement of five degrees-of-motion of a measurement object; Figures 13(a) and 13(b) respectively illustrate, a binding cell geometry and an associated line illumination geometry for use for performing measurements of molecular surface binding; Figures 14(a) and 14(b) respectively illustrate illumination, for the purposes of performing label free binding measurements, of: (a) a flow cell in a reference state in which there is no surface binding; and (b) a flow cell in a bound state in which there is surface binding; Figure 15 shows an interferometer apparatus for performing label free binding measurements; Figure 16 illustrates one configuration in which a dual spot configuration can be used in conjunction with a surface plasmon resonance (SPR); Figure 17 illustrates another configuration in which a dual spot configuration can be used in conjunction with a surface plasmon resonance (SPR); Figures 18 illustrates how the interferometer apparatus may be adapted for application in interferometric flow cytometry for transrnissive measurement; Figures 19 illustrates how the interferometer apparatus may be adapted for application in interferometric flow cytometry for reflective measurement; and Figure 20 illustrates, in simplified form, the basic interferometer output that results from passage of a particle during the interferometric flow cytometry illustrated in Figures 18 and 19.

Overview Figure 1 schematically illustrates, in overview, a general configuration of exemplary interferometer apparatus, according to one embodiment, generally at 10. The interferometer apparatus comprises collimation optics LO, lenses L1 to L5, shearing optics SO, detection optics DO and a signal processor SP.

The collimation optics LO comprise an illumination source P for producing the electromagnetic waves used by the interferometer apparatus 10, and lens L1. In this exemplary embodiment, the illumination source P comprises a single mode fibre pig-tailed monochromatic source such as a laser diode or Super Luminescent Emitting Diode (SLED). The light from the illumination source P is collimated by the lens L1 to form a collimated ray pencil (only the central beam of which is shown for simplicity) before entering the shearing optics SO.

The shearing optics SO, in this embodiment, comprise a Michelson configuration (shown in more detail in Figure 2). The shearing optics SO divide the beam into two component beams 1, 2 which diverge at an angle ±a to the optical axis (small enough for the small angle or paraxial' approximation to apply) from a common point 0 until the diverging light reaches second lens L2, located at a distance 12 from the common point 0.

The lens L2 causes the two component beams 1, 2 to converge, at an angle ± a' to the optical axis (small enough for the small angle or paraxial' approximation to apply), to conjugate point 0' at a conjugate distance 12' from lens L2. Lens L2 forms, at conjugate point 0', an image of the light at the common point 0, with magnification m1=121/12. Translated images of the source P are thereby formed at points P1 and P2 in the focal plane of L2 at the focal length f2 of lens L2, and are symmetrically centred at a separation s about the central optical axis where s=±f2a(using the small angle approximation).

Lens L3, having focal length f3, is located at a distance 13 from the focal plane of lens L2 and receives light from it as illustrated in Figure 1. Lens L3 is arranged at a distance 13' (where 13' = (f3'-13')') from an object plane D (e.g. a plane of a surface of a measurement object) such that the object plane D is at the plane conjugate to the plane containing P1 and P2, with reference to lens L3. An image of the translated images at P1 and P2 is thus formed at points P1' and P2' in the object plane D, by the lens L3, with magnification m2=131/13 and centred with separation ± s<' = m2sX from the optical axis. Two discrete regions of the object are thereby illuminated with mutually coherent light fields or spots' centred at points P1' and P2'. The light fields projected onto the measurement object produce an associated speckle pattern (where the surface on which the light is projected is optically rough) for observation via the detection optics DO. Moreover, interference between the light fields projected onto the measurement object produce a fringe field at the detection optics DO.

The radius of each illumination field produced by lens L3, centred respectively at P2' and P11, is a combined function of the optical parameters for the layout shown in Figure 1 and the form of the illumination source P. In this embodiment, the illumination source P is assumed to generate, via L1, a collimated beam profile having a lie2 radius w1. The radius wi,, of each illumination field centred respectively at P2' and P1' is then given, using standard Gaussian beam propagation, by: = 0.32 m2Af2 (1) ITwi where Xis the wavelength of the light.

The distance between P21, P1' is 24 where, = (2) The detection optics DO (shown in more detail in Figure 3), in this embodiment, comprises a photo detector PD and lenses L4 and L5. In order to make a measurement the objective speckle pattern, from the illumination regions at P1' and P2', at an entrance pupil of the detection optics DO is imaged onto the plane of the photo detector PD by means of lenses L4and L5.

The signal processor SP receives data representing the light incident on the photo detector PD and processes it to derive information identifying characteristics of the surface of the measurement object onto which the light is projected in the object plane D. Beneficially, therefore, it can be seen that the interferometer apparatus 10 uses beam shearing optics SO to project two mutually coherent areas of light onto an object at P1' and P2' via a common path, thereby making the interferometer intrinsically robust.

Further, the interference between the projected areas forms a carrier fringe field, at the detection optics DO, with the phase of the fringe field being determined by the difference in the optical path length of the two sheared beams to the object.

Beneficially, therefore, by measuring changes in the phase of this fringe field it is possible to determine changes in the relative path length as caused by changing surface parameters caused, for example, by movement of the surface as a result of flexing or vibration.

This carrier fringe field may beneficially be observed in the presence of speckle pattern thereby enabling the interferometer to be used for the measurement of objects with either optically rough or smooth surfaces.

In addition, because, the path lengths of the interfering beams are matched short coherence sources such as SLEDs may be used. These have non-resonant emission and are not subject to modal phase noise characteristic of standard multi-mode lasers sources. The short coherence also has the knock on benefit of effectively eliminating multiple path interference that can result from the use of a single mode laser which has an intrinsically long coherence length The above features, combined with the use of either carrier fringe phase quadrature or tracking algorithms, also provide the basis for designs, described in more detail later, for which optimal performance may be achieved for a wider range of applications and operating environments than conventional interferometry allows.

Shearing Optics The beam shearing optics SO will now be described in more detail, by way of example only, with reference to Figure 2 which shows beam shearing optics, based on a Michelson interferometer, that are suitable for use in the interferometer apparatus 10 of Figure 1.

In the arrangement shown in Figure 2, a pair of Michelson mirrors M1 and M2 and a beam splitter BS are arranged with the mirrors ML M2 inclined at ± a /2 to form the two beams diverging from 0 via the beam splitter BS at ± a/2 to the z axis (as shown in Figure 2).

Sinusoidal modulation SM of the phase in one arm of the Michelson interferometer may be introduced by applying a small sinusoidal displacement normal to the surface of a mirror (in this example M1) in the Michelson interferometer using an actuator A (such as a piezo stack or the like) attached to the mirror M1.

Detection Optics The detection optics DO will now be described in more detail, by way of example only, with reference to Figures 3(a) and 3(b) which show, in xy and xz planes respectively, detection optics DO that are suitable for use in the interferometer apparatus 10 of Figure 1.

In the detection optics DO of this embodiment, the photo detector PD is a linear photo detector comprising a linear array of individual detectors such as photodiodes, lens L4 comprises a spherical lens arranged, at the entrance pupil of the detection optics DO, to form aperture A at which the light field diffracted from the measurement object is received. Lens L5 comprises a positive cylindrical lens. As seen in Figure 3(a), the lens L5 is arranged such that, in the yz plane, it does not affect the passage of light through it.

The linear photo detector PD is arranged parallel to a line containing points P1' and P2' (e.g. along the x axis) and the plane containing points P1' and P2' is imaged onto the linear photo detector PD, along the x axis, by the spherical lens L4 (as seen in Figure 3(a)).

As seen in Figure 3(b), the lens L5 is arranged such that the objective speckle pattern is imaged onto the photo detector PD, in the long axis of the linear photo detector, by the positive cylindrical lens L5. In this axis lens L4 serves to gather light onto lens L5, thereby lowering the numerical aperture (NA) required for lens L5.

The resulting image A' is an image of aperture A along the x axis, and of the object plane D in the y axis. This arrangement maps all of the light passing from points P1 and P2 through A onto the linear PD, and maintains the elevated content at the spatial frequencies corresponding to the fringe spacing xp (see equation (5) below).

In the detection optics DO, both axes are focussed by ensuring: 14 = 15 + 15 (3) Where 14' is the distance from lens L4 to the plane conjugate to D for lens 1.4, and 15 and 15' are the respective distances from lens L5 to each of its imaging conjugates in the xz plane as illustrated in Figure 3(b).

Under these conditions an image is formed at points P1" and P2", of the object plane focal spots at points P1' and P2', is formed at a distance l" from L5, centred with a separation ±s<" about the central optical axis, with: = s(1-lp) (4 rn3!4 where the magnification provided by lens L5, m3 = l'/l.

The two beams diverging from P1" and P2" interfere in the photo detection plane to generate fringes of spacing Axe, with: AXF = () 2 s where Xis the wavelength of light.

When D is optically rough L5 will also image the objective speckle pattern present in the plane of the aperture A. This speckle pattern will, however, be modulated by the fringes described above. This speckle pattern will have an average dimension x5 given by: = rn11A (6) WI,, where wi,, is the radius of illumination at P1' and P2' (see equation (1)).

Unlike conventional speckle pattern interferometry, imaging is of the objective speckle pattern rather than the subjective speckle pattern. Unlike conventional speckle pattern interferometry, therefore, the average speckle size for a given wavelength is defined by the dimensions of the illumination field rather than by the characteristics (such as the f-number) of the viewing optics, as would be the case for subjective speckle.

Figure 4 shows an exemplary representation of how, for optically rough surfaces, carrier fringe field may be superimposed on a speckle pattern in a situation where the average speckle size Ax is larger than the fringe spacing Axe. The ratio n5f of the average speckle size Ax to fringe spacing AXF in the detection plane is given by: (7) The optical system may thus be designed such that nsf >1 thereby enabling the fringe pattern to be observed within the individual speckles as shown in Figure 4. The observation of the carrier fringes in this way beneficially enables the interferometric measurement to be extended to optically rough surfaces.

It will be appreciated that whilst the above example has been described with reference to a 1D (linear) photo detector, the design may be extended to a 2D detector array by replacing the L5 cylindrical lens by an equivalent spherical lens, albeit that this would change the required processing scheme, and would generally reduce the achieved signal to noise ratio.

Moreover, whilst having the detection optics DO and the optics for illuminating the measurement surface separately is advantageous as it allows analysis to be carried out remotely from the illumination apparatus, it will be appreciated that in some applications it may be advantageous to have the detection optics DO integrated within the main illumination apparatus (e.g. as shown in Figure 15).

Operation to measure movement of measurement object Figure 5 illustrates, in simplified form, the principle of operation of the interferometer to measure rotational movement of a surface of a measurement object.

As seen in Figure 5, a rotation of the surface of a measurement object, at the object plane D, through an angle AO around they axis (perpendicular to the plane of Figure 5) through the mid-point 0 between P1' and P2' introduces a relative phase difference of Ap = 4irs'AO/X between the two beams. This translates the speckle pattern at the aperture plane A by a distance 2A0l4.

The phase change due to rigid body displacements (dx. d, d), and in plane rotations and tilt about the x axis are common to both beams and so do not create a relative phase change. Similarly, higher order surface motion (e.g. a flexure of the surface which leaves the midpoints of P1', P2' unchanged) alters the speckle structure, but do

not translate the underlying fringe field.

The common object illumination therefore enables either small angular tilts about a point in the surface or the relative refractive index at the proximity of P1', P2' to be measured in the presence of macroscopic rigid body displacements, macroscopic movement of the sensor, and refractive index variations common to the beam paths and enhances the intrinsic robustness of the interferometer.

In the case of optically rough surfaces, however, such macroscopic displacements will result in the speckle pattern decorellation of the carrier fringe field and the maintenance of continuous phase measurement under these conditions is a particularly beneficial aspect of the signal processing used to extract information about the measurement object, as described in more detail below.

Signal processing to determine changes in rotational position Operation of the signal processor SP to determine a change in rotational position will now be described in more detail, by way of example only, for the photo detector PD comprising a linear array as described in the above embodiment, and for a photo detector PD comprising a point detector (e.g. an individual photo diode or the like).

Linear Array For linear array detection, a linear array having a pixel height greater than w,l41/l4 is used at the photo detector to ensure that the light from the measurement object is all collected at the sensor. The pixel pitch of the linear array is approximately Ax/4 (or possibly lower) thereby allowing a sufficient fringe resolution.

Figure 6 shows an exemplary spatial power spectrum of a one dimensional (1D) sensed image provided by a linear array for an optically smooth or specular' surface (shown as a solid line) and an optically rough surface (shown as a dashed line). In Figure 6, the discrete spatial power spectrum of the sensed 1D image produced at the linear array is the autocorrelation of the complex amplitude function at the illuminated surface of the measurement object.

The elevated content around the spatial frequency WF corresponds to the fringe spacing Ax in reciprocal space; this region arises from one of the spots of light incident on the measurement object interfering with the other, and is referred to herein as the fringe content or fringe region. The area around the origin results from the self-interference of each spot, which is referred to herein as the speckle content or speckle region. Configuring the optics of the interferometer apparatus such that the separation s,' between each region of illumination on the surface of the measurement object and the central optical axis is much greater than the radius of the illumination w, (s >> wv,) ensures that the fringe and speckle regions are well separated.

The processing algorithm compares the complex spatial spectra (obtained via a discrete Fourier transform (DFT)) of two consecutive 1D images or frames'. A pure rotation of the object AO results in a linear phase difference between the two frames in reciprocal space, with a gradient proportional to AO. Whilst confounding factors can result in a deviation from this linearity for the speckle content, the cancellation of these factors between spots means that it provides a sufficiently accurate model for the fringe content.

The phase gradient in the fringe content can be determined using linear regression; weighted by the power in each spatial frequency (the weighting being selected to additionally remove the speckle content). From this the rotation of the object A9 between the two frames can be determined.

The above method is applicable when the rotation of the object M is less than half the fringe spacing divided by the distance from the measurement object to lens 14 (A8 < Ax/2l4) (i.e. the x translation of the fringe field is under half a fringe). If this is not the case then the integer number of fringes translated between frames is determined first, for which the signal inclusive of the larger scale speckle structure can be tracked in the same manner as is described above for the fringe content only.

However, as any approach for doing this could be susceptible to errors at integer multiples of Ax this can be done most successfully where the bulk motion is at a frequency far lower than the frame rate. The integer number of fringes shifted per frame can then be averaged over many frames, and the sub-fringe shift then calculated using the methods described.

Where this averaging technique is used, it is important that the individually calculated frame-to-frame shifts have zero mean error. For this reason standard phase correlation techniques may not be suitable. One approach found to be particularly successful is to find the integer pixel translation which minimises the sum of the squares of the pixel errors.

Point Detector In the case of a point detector, the point detector measures the total intensity is some region of the fringe field at photo detector PD. Rotations of the measurement object result in an output F. which is sinusoidal (plus some constant) as the fringe field sweeps past the detector. Determining changes in phase &p of this sinusoid is therefore effectively equivalent to measuring the rotation M. The sinusoidal content of this signal is maximised when the width of the point detector is equal to half the fringe spacing (i.e. AXF/2).

Phase generated carrier demodulation is then use to extract the rotation AO from this oscillatory output. This is achieved by introducing a known additional phase modulation AQ irsin(wt) into one of the arms of the Michelson interferometer shown in Figure 2 to introduce a known sinusoidal variation to the angle at which the sheared component beams 1, 2 diverge from the interferometer at Q. The piezo actuator A attached to one of the Michelson shearing optics mirror, for example M1 as shown in Figure 2, may be used for this purpose. The signal at the detector then takes the form = sin(msin(at) + q0) (8) where q31 is the phase of the fringe field whenAq = 0.

The amplitude of the fundamental and second harmonic of are in quadrature as a function ofw0. This means that the phase q can be determined unambiguously, and bulk motions covering multiple fringes can be tracked.

The quadrature relationship holds provided that q is approximately constant over the course of a single modulation cycle. For this reason signals can only be detected using this processing scheme at a frequency lower than w/2 and with the rotation AO being much less than the separation s,<' of the each region of illumination from the central optical axis multiplied by the frequency of the additional phase modulation component divided by the wavelength of the light (AG << ws/A).

Signal processing to determine tangential translations Operation of the signal processor SP to determine tangential translations (specifically in-plane movement of the illuminated surface of the measurement object in the x direction) will now be described, by way of example only, with reference to Figure 7 which illustrates the effect of a tangential translation of a measurement object on the speckle envelope and fringe patterns for that object.

In the example of Figure 7, the movement represented is a pure' translation (with no rotation of the measurement object) along a straight line containing the spots P1' P21. Hence, the phase of the fringes in the fringe pattern remains unchanged whilst the speckle envelope moves as illustrated in Figure 7.

Determination of the extent of the tangential translation can be achieved by defocussing the projection optics (Figure 1) such that the beam waists for spots P2', P1' are formed an axial distance ZR from the object, where ZR Is the Raleigh range for P2', P1' thereby maximising the wavefront curvature R of the two beams at the object.

Assuming that the object is an optically rough surface with profile f(x) then the complex amplitude EN) for a single spot upon reflection from the surface of the measurement object is given by: E(x) cc exp L -ik (x2 + 2f(x))j (9) where k is the wavenumber of the light and i is the square root of -1.

If the measurement object is translated a distance Sx parallel to the line containing spots P2', P1' then the new amplitude, E' (x), is: 2x2 /x2 E'(x)ccexp -2-ik-+2f(x-Sx) Wp' 2R 2(x-Sx)2 4xSx ccexp -2 + 2+O(6x2) Wp' Wp' f(x-flx)2 (x-Sx)öx ZR + R +Zf(x-Sx)+const /4x8x\ xsx E(x -ox) x exp x exp (-ik -) (10) The first term represents a pure translation of the field at the object, resulting in a linear phase shift of the light along the senor, which is not detectable. The second term is surpressed bt the first, except for where x -war, so is of order exp (_±_) 1, assuming--<<1. wP,

It can be seen, therefore, that the result of the translation is results from the third term, an apparent linear phase shift across the spot, proportional to the size of the translation öx.

This also applies for the other spot so that each spot receives an identical linear phase shift. These two shifts cancel out in the fringe region (see figure 7) but appear as a translation of the speckle component at the sensor.

This phase shift can thus be measured, using the techniques described above for measuring the phase shift of the fringe field using the linear array, and hence the magnitude of the translation of the measurement object in the x direction can be determined. In the event that the measurement object is exhibiting rotation as described earlier in addition to the tangential translation, the phase shift contribution made by such rotation can determined from the changes to the fringe field (as described earlier) and subtracted from the measured phase shift effectively to eliminate the effect of the rotation on the measurement of tangential translation.

It will be appreciated that, via the inclusion of a second orthogonal spot-pair, using this technique allows translations tangential to the surface to be measured along either of two axes within the plane of the measurement surface. Further, rotation of the measurement surface about an axis normal to the plane of the measurement surface can be determined by measuring the relative differential translations at two separate locations Modifications and alternatives A detailed embodiment has been described above. As those skilled in the art will appreciate, a number of modifications can be made to the above embodiment whilst still benefiting from the inventions embodied therein. By way of illustration only a number of these alternatives and modifications will now be described.

Simplified Beam Shearing optics Figure 8 shows another example of shearing optics SO that may be used to generate two component beams for the interferometer apparatus of Figure 1 (or other configurations of interferometer apparatus described herein or otherwise). The beam shearing optics SO of Figure 8 simplifies the shearing optics SO compared to those based on the Michelson interferometer of Figure 2.

As shown in Figure 8, the shearing optics SO comprise a beam splitter BS and a bi-prism BP.

The beam splitter BS is arranged, at an angle relative to the main optical axis, to generate the two parallel component beams Al, A2 from a collimated beam produced at lens LA1 via lens LA2.

The bi-prism BP is arranged to receive the parallel component beams Al, A2 and to converge the two component beams Al, A2 to a common point of intersection (corresponding to Q' in Figure 1). Lens [Al and lens LA2 are adjusted to create the focal points at PAland PA2 as required.

In this example, sinusoidal plane modulation SM may be created by applying a lateral sinusoidal displacement SM to the bi-prism BP via the actuator A as shown in Figure 8.

It will be appreciated the above simplified system may also be configured to create a pair of collimated beams that diverge from a point corresponding to Q in Figure 1 (in a similar manner to the shearing optics SO based on the Michelson interferometer of Figure 2) with lens LA? following ci and being arranged to modify the component beams Al, A2 as described with reference to Figure 1.

Scanned Beam Optics The optical configuration of the interferometer apparatus described with reference to Figure 1 enables measurement to be made at a fixed point in the object. Another embodiment will now be described with reference to Figures 9 and 10, by way of example only, in which measurements may be made over a range of positions on the object.

Figure 9 shows, generally at 90, another configuration of interferometer apparatus that may be used to enable the point of measurement, as defined by the centroid of the dual spot illumination, to be scanned over the measurement object thereby enabling measurements to be performed sequentially over a two dimensional (2D) surface. Figure 10 shows part of the configuration shown in Figure 9 and illustrates the scanning operation of that configuration.

The interferometer apparatus 90 of Figures 9 and 10 comprises a plurality of lenses [BI, [B? and [B3, a beam splitter BS, and a scanning' mirror M5.

Referring to Figure 9 in particular, collimation optics (not shown) produce a beam of collimated light which is sheared, using one of the configurations of shearing optics (50) described previously, to produce two component beams Bi, B2 that each diverge at an angle ± a to the optical axis, from the shearing optics SO at common point 0, to the lens LB1. From lens LB1, the two component beams B1, 32 propagate, parallel to the optical z axis.

The lenses LB1 and LB? have respective focal lengths fBi and fB2, and are arranged to have a shared focal plane through PB1 and PB2. The two component beams B1, B2 travel via the focal points at PE1 and PB?, each propagating in a direction parallel to the optical z axis with a separation of ± f91a8 relative to this axis (using the small angle approximation). The beam splitter BS is arranged such that the component beams Bi, B2 from lens LB1 pass through it, essentially unhindered, to lens LB2.

The lens LB2 and the scanning mirror M5 are arranged such that the rear focal plane image of the component beams Bi and B2 is incident on scanning mirror M. The mirror M5 is inclined at a variable angle to the optical axis although, in Figure 9, it is shown at an angle of 45° to the optical axis which, in this embodiment, is its neutral position.

The image incident on the mirror M5 corresponds to a plane in which the collimated light from PBT and PB2 overlap (as seen in more detail in Figure 10). This results is two plane wavefronts centred at ci' that diverge at an angle a' (=f32a8/fBl) relative to an optical axis perpendicular to cia'.

Lens LB3 is an F/U' (also known as an f/theta scanning') lens centred on this axis perpendicular to 0.0', at its working distance dB3 relative to Q'. Lens LB3 transforms the incident plane wave front into two focal points P'Bl and P'2 incident perpendicular to a surface of a measurement object placed in the focal plane of lens LB3 (at its focal length fB3) and separated by a distance 2f33as'. Light reflected from the surface of this measurement object is coupled back to the detection optics via the scanning mirror M5 and the beam splitter BS placed between Lenses LBland LB2.

In operation, therefore, the variation in the angle of the incidence on the scanning mirror M, in response to a time varying scan angle U(t), causes B1 and B2 to be either continuously or step scanned across the object over an area 2fB3Ox by 2f338y.

Under these conditions phase measurement synchronous with the scan enables a 2D image of differential phase variation to be created, for example using the signal processing methods described earlier.

Detection Optics Whilst the detection optics configuration illustrated in and described with reference to Figure 3 provides a particularly beneficial configuration in terms of the simplicity with which it provides the required imaging properties, the measurement techniques described for use with the detection optics of Figure 3 require that, in the xz plane (Figure 3(b)), the photo detector PD contains a near diffraction limited image of A, with as little distortion as possible. Conversely, in the yz plane, it is only necessary for substantially all of the light passing through the aperture A at a given y coordinate to be condensed onto the height of a pixel.

Figure 11 shows another arrangement of the detection optics DO, which take advantage of the availability of high quality imaging lenses, to optimise the configuration of the optics. Figure 11(a) shows the configuration in the yz plane and Figure 11(b) shows the configuration in the xz plane.

As seen in Figure 11, the detection optics DO comprise lenses Lc4, L5 and Lc6.

Lens Lc4 comprises a spherical lens and is arranged in a similar manner, relative to the object plane, as lens L4 in Figure 3. Lens Lcs is a diverging cylindrical lens arranged with conjugate points in the yx plane at the measurement object and at the aperture A at lens Lc4 (i.e. at the focal distance of 1C5' from lens L5). Lens Lc5 causes the light incident on it to diverge in the yz plane but not in the xz plane.

Lens L6 is a so called well corrected' multi-element imaging objective lens arranged to image A onto the photo detector PD, with the spherical lens Lc4 gathering light onto it. The lens L4 has a back focal distance 1c4' equal to the front focal distance 1c6 of lens Lc6. The lenses Lc4 and Lc6 and the photo detector PD are arranged such that lens L4 is at a distance equal to 1C4'/ 1c from lens LcG and photo detector PD is at a distance from lens Lc6 that is equal to the rear focal distance 1c6' of lens L6.

As seen in Figure 11(a) lens L6 is arranged to converge the light that it receives via lens L5, from aperture A onto the photo detector PD (e.g. a linear photo detector as described previously). The linear photo detector PD is arranged along the x axis, and the plane containing points P1' and P2' (Figure 2 refers) is imaged onto the linear photo detector PD, along the x axis.

As seen in Figure 11(b), the lenses Lc4 and Lc6 are arranged such that the object plane is imaged at Lc6,and the objective speckle pattern is imaged onto the photo detector PD, in the long axis of the linear photo detector.

Like Figure 3, therefore, the resulting image A' is an image of aperture A along the x axis, and of the object plane Din they axis.

Whilst the detection optics configuration illustrated in and described with reference to Figure 3 provides a particularly beneficial configuration in terms of the simplicity, the piano-convex cylindrical lens used to do the imaging of the objective speckle pattern can exhibit associated aberrations that limit performance through, e.g. distortion making a translation appear to be a translation plus stretch, instead of a pure translation. There are, however, many off-the-shelf spherical lenses which image without these aberrations. A configuration, such as that described above, which uses a spherical lens to image the objective speckle pattern can, therefore provide greater flexibility and improved results.

It will be appreciated that there are multiple possible detection optics configurations for detection optics which image the object plane at some point in front of the sensor (e.g. the plane of P11' and P2" as pictured in Figure 3).

Providing Additional Motion Sensitivity It is also possible to provide additional motion sensitivity, compared to earlier examples, by providing a system which illuminates the object with more than one pair of spots, thereby providing sensitivity around other axes.

Figure 12, for example, shows a four-spot interferometer system which can provide sensitivity to 5 degrees of motion.

In the system of Figure 12, four spots of light are provided on the measurement object (e.g. using an appropriately adapted version of the optics described with reference to earlier Figures).

The 4 different spot pairs are then spatially filtered (e.g. using suitably positioned beamsplitters and slits) to pick out separated pairs of spots such that from each specific spot pair a different rotation and translation measurement may be derived.

Considering the spot pairs as labelled in Figure 12, for example, we can calculate these 5 degrees of motion as follows: = (e13 + = (e12 + = [(d12 -d34)/S + (d24 -d = (d12 + d34/2 d=(d13+ d24)/2 Where: emn signifies the rotation and dmn signifies the translation as obtained from taking a measurement using spots Sm and Sn. 6,<, O, and 6 respectively signify the calculated rotation around the x, y and z axis d, d, d, respectively signify the translation in-the-direction-of the x, y and z axis.

Linear Illumination It will be appreciated that the scanned beam optics described with reference to Figures 9 and 10 enable measurements to be made at multiple locations. Figure 13 illustrates a linear sensing scheme which allows measurements to be made, at multiple locations substantially simultaneously, in a specific practical application (measurement of variations in refractive index due to molecular surface binding).

Specifically, Figures 13(a) and 13(b) respectively illustrate, a binding cell geometry and an associated line illumination geometry for use for performing measurements of molecular surface binding.; In the configuration of Figure 13, two lines of illumination are imaged onto a measurement object as illustrated in Figure 13(a) using apparatus similar to that described with reference to Figures 9 and 10 to generate sheared beam components Dl and D2 and project them on the surface of the measurement object.

Measurements can be derived from the lines of illumination using detection optics similar to that illustrated in Figures 3 or 11, or any suitable variation thereof, but using a two dimensional photo detector PD array (in the xy plane) as opposed to a linear detector (in the x direction only). Each row of the 2D photo detector can be processed in the same manner as for the linear detector, but with the y coordinate across the detector having a direct correspondence to the y coordinate at the object.

As will be described in more detail later with reference to a particular application in which this approach is particularly useful, in this configuration a number of sites on the object (B12...) can be designated for inspection. These inspection sites B12* may be compared not only to a local reference site (R1,2...) but also to a neighbouring pair of reference sites (R1112...1,R21 22 2n). This allows for the effect of any bulk rotations of the substrate effectively to be removed.

Various other modifications will be apparent to those skilled in the art and will not be described in further detail here.

Applications It will be appreciated that the interferometer apparatus described herein has benefits in many applications. A number of these applications will now be described by way of example only.

The applications fall into two main areas: (a) the remote measurement of the motion of optically rough objects; and (b) the measurement of small variations in the refractive index due to molecular surface binding.

Remote motion measurement There is an established industrial requirement for differential vibration measurement, e.g. in the field of automotive component testing. Currently this requirement is addressed using an approach that requires two separate measurements from two locations (typically each using laser Doppler vibrometry) and compares these.

In contrast using the interferometer apparatus described herein, an interference pattern is created between the returned light from two locations, and capture the differential motion from single measurement, as described above. As well as simplifying the measurement, this also removes the effect of many confounding factors and significantly improves measurement accuracy.

In addition to differential vibrations, the apparatus and methods described herein allows measurement of any translational motions of the object being measured.

Whilst devices which can track the translations of moving objects are available commercially, these require a specific target (e.g. retro-reflective prism) for tracking, whereas the apparatus and methods described herein allow measurement of the motion of any rough surface, using the laser speckle from the surface roughness as a reference.

In combination the apparatus and methods described herein enables a single motion measurement system capable of measuring differential vibration around two axes, macroscopic translations in a plane normal to the optical axis, and rotations around the optical axis. It will be appreciated that the measurement capability could be further extended to provide the addition of accurate distance measurement (e.g. using time-of-flight) to enable remote measurement of the full 6 degrees-of-motion (using the apparatus of Figure 12).

Such a system can measure distances up to 105 of meters or even greater subject to laser safety imposed limitations.

General Concept The general concept for measurement of molecular surface binding is illustrated in Figures 13 to 15.

Figures 13(a) and 13(b) respectively illustrate, a binding cell geometry and an associated line illumination geometry for use for performing measurements of molecular surface binding.

Figures 14(a) and 14(b) respectively illustrate illumination, for the purposes of performing label free binding measurements, of: (a) a flow cell in a reference state in which there is no surface binding; and (b) a flow cell in a bound state in which there is surface binding.

In the unbound state (Figure 14(a)) the beams Dl and D2 are incident on a binding site B and reference site R respectively (e.g. at a binding site B12* and associated reference site R12. shown in Figure 13(a) where the scanning configuration of Figures 9 and 10 is used) on the internal face of an optically transparent substrate S that forms part of a flow cell FC.

Referring to Figure 14(b), in operation fluid containing molecules M is passed through the flow cell. Molecules with appropriate affinity become bound to the binding sites B resulting in the formulation of a cavity of thickness t at the substrate local to this site. This increases the optical path length of component beam Dl relative to component beam D2 by 2nbt where the cavity thickness t will depend on the extent of the binding and nb is the refractive index of the bound molecules. The resultant phase shift of beam Dl relative to beam 2 (=4lrnbt/A) is measured by the interferometer.

For this application a scanned configuration of interferometer, similar to that described with reference to Figures 9 and 10, is preferred because this has normal surface illumination and may be extended for measurement at multiple sites using the scan mechanism. In such a system, for example, the binding site element (such as the flow cell FC) is placed in the object plane D shown in Figure 9.

Figure 15 shows an interferometer apparatus, for performing label free binding measurements, that incorporates a scanned configuration, similar to that described with reference to Figures 9 and 10, generally at 150. In the interferometer apparatus shown in Figure 15 a binding site element of a flow cell FC is located in the object plane D. A set of binding sites B and reference sites R, in the binding cell configuration shown in Figure 13, are illuminated by respective lines of illumination from component beams Dl and D2, as shown in insert isa. The shearing optics, 50, operate as previously described, sending a pair of sheared, collimated beams to the elements lens LD2, lens LD3, mirror M5 and lens LD4 which operate in a scanning configuration similar to that shown in Figure 9, although lens LD4 in this example is a cylindrical lens configured to produce a line focus. The lines of illumination are measured, using detection optics DO which receive illumination returned from the flow cell via beam splitter B2. DO, in this example, consists of two perpendicular cylindrical lenses: lens LD5 which is configured to image the object plane, D, onto a 2D photo detector PD in the y-axis; and lens LD6 which is configured such that, in the x-axis, the PD is in the Fourier plane of the reimaged lines at D'. The detection optics produce interference patterns corresponding to each binding site B12. and associated reference site R1,2* (and corresponding to each neighbouring pair of reference sites R11121 R21222) at the 2D photo detector, as shown in insert lSb. The fringes in these patterns move in dependence on relative changes of refractive index at the binding site due to the associated phase changes.

The patterns corresponding to each binding site B12. and associated reference site R12. will also vary with changes in phase associated with bulk rotations of the measurement object. However, because the pattern for the neighbouring pair of reference sites R11121,R21 22 2n will also exhibit this phase change (but will not exhibit changes due to changes in refractive index), the effect of bulk rotations can be eliminated by comparing the variation in pattern associated with each binding site B12. and associated reference site R1,2. with any variation in the pattern associated with the neighbouring pair of reference sites R11 12,R21222.

Surface Plasmon Resonance (SPR) Figures 16 and 17 each illustrates a configuration for using a dual spot configuration, as described previously, in conjunction with a surface plasmon resonance (SPR) illumination geometry for ultra-high sensitivity, phase domain label free detection.

In each of Figures 16 and 17 a prism 160, 170 is provided having a resonant surface CS (in these examples, the resonant surface CS is a gold surface of a given thickness) in a manner suitable for conventional SPR as those skilled in the art would readily understand. In operation, the prism 160, 170, may be arranged on a flow cell for which the molecular binding measurements are to be performed.

A pair of parallel component beams El and [2, Fl and F2 are produced via the shearing optics SO (e.g. from a collimated beam generated from an illumination source using an optical configuration described previously). The component beams El, [2, El, F2 are directed through prism 160, 170 to illuminate the resonant surface CS, that is provided on the face ab' of the prism 160, 170 via a lens L[3, L3, at an angle 13 to the normal of the gold surface CS. The apparatus is arranged such that the angle 13 corresponds to the angle required for resonant interaction with the given gold coating thickness.

In the apparatus of Figure 16, the component beams [1, [2 as reflected by the resonant surface CS are received and detected by detection optics DO that are separate from the shearing optics SO. Contrastingly, in the apparatus of Figure 17, the component beams Fl, F2 as reflected by the resonant surface CS are incident on a mirror M arranged to reflect the component beams back towards the resonant surface CS and, ultimately, detection optics DO which are combined, in a single optics configuration with the shearing optics SO.

The differential phase between the component beams, resulting from the effective lengthening of one component beam relative to the other associated with binding at different sites within the resonant surface CS, can then be measured at the detection optics DO as described previously.

The configurations shown in Figures 16 and 17 may each be operated in a scanned mode by taking into account the fact that the beam waist at P1and P2 must accommodate the varying ratio of glass to air and depth of field required for the nominally 45° angle of incidence as the beam is scanned. Specifically, referring to Figure 16, if the spots are scanned along the line of the prism surface ab, whilst keeping the beam at 45° to the line of the prism surface ab, then the light has to travel through more glass to get to the object plane at b than it does at a, meaning that the light comes into focus too soon. Accordingly, in the configurations shown in Figures 16 and 17, the spots are provided with a large enough depth of field to be sufficiently in focus at the object at both ends of the scan. This problem may also be mitigated, for extended fields of view, by translation of the prism and/or the optics in a direction PQ parallel to the prism surface ab, whilst maintaining the angle j3 at the required resonant angle.

Flow cytometer configurations Figures 18 and 19 each illustrate how the interferometer described herein may be adapted for application in interferometric flow cytometry for transmissive and reflective measurement respectively.

In arrangements of Figures 18 and 19, focal points P1' and PG2', PHi' and PH2' are formed at the centre of a flow cell FC through which optically transparent particle 0 of radius rq and refractive index nq are carried at a flow velocity vf parallel to the x axis by an optically transparent fluid of refractive index n1.

Figure 20 illustrates, in simplified form, the basic interferometer output that results from the passage of the particle 0 through the focal points PG1' and PG/, PHi' and H2 (provided the particle diameter 2rq is less than the beam separation).

In Figure 20, the signal is correlated with the position of the particle at the specific positions pi to P6.

When it is assumed, for simplicity, that the interfering beams P1' and P2' or PHi' and PH2' have the same intensity 112=11=12 then the intensity of the two beam interference is ld(t) is given by: 1(t) = 2112 (1 + cos(cq + /.Xq)) (11) where c/q is the phase of the fringe field at the detector in absence of a transiting particle, and is the phase change generated by the particle transition, i.e.: = Nmrq(nq-nj) (12) forrq >w,and: -IVWTq3(flqflf) 13 -2iwp,2 otherwise.

In both cases N = 2 for transmission, N = 4 for reflection.

If we chose cbq ir/2 then equation 11 reduces to the form Ia(t) = 1+ K acbq (14) where bo2h1z(1+q7) K = 2 12 Hence, LWJq = 1d(t) Jo (15) Equation 14 defines the time varying interference signal ld(t) shown in Figure 20 and is a combined function of the particle size rq and refraction index nq. If the flow velocity vf is known, then the particle size may be determined from separation in time Atnm = tm -t, between the signals observed at the particle position at various combinations of position: = 2 2 -wi,' = -2 +w,) =2s-At25v1 (16) It will also be recognised from the above analysis that the signal ld(t) defines the convolution between the particle size as defined by its refractive index profile and the PG1' and PGZ' or PHI' and PH2' illumination structure. Analysis of the detected interference signal ld(t) based on the above and in accordance with equations 12, 14 and 15 thereby provides a means by which the particle size and refractive analysis may be measured effectively.

Summary of biological applications of surface binding measurement A number of specific applications in which the surface binding measurement, using the interferometric apparatus described herein, may be used in specific applications will now be described by way of example only.

Nucleic acid testing Immobilised, sequence specific probes for nucleic acid can be arranged at defined locations to act as bait for specific nucleic acids. Following the exposure of nucleic acids to these probes the binding of specific nucleic acids can be quantified by examination using the interferometer apparatus and/or interferometry methods described herein.

Protein testing Immobilised, sequence specific probes for protein can be arranged at defined locations to act as bait for specific proteins. Following the exposure of proteins, or parts of proteins to these probes the binding of specific proteins can be quantified by examination using the interferometer apparatus and/or interferometry methods described herein. This could be used to evaluate the protein content of a sample which is being analysed on the array or the affinity of different probes to specific proteins.

Evaluation of proteins and nucleic acids on a single array Immobilised, sequence specific probes for proteins and nucleic acids can be arranged at defined locations to act as bait for nucleic acids and proteins in the same sample; enabling both proteins and nucleic acids to be evaluated at the same time from the same sample. Following the exposure of nucleic acids and proteins to the probes the binding of specific nucleic acids and proteins can be quantified by examination using the interferometer apparatus and/or interferometry methods described herein.

Cell evaluation Whole cells or fragments of cells could be captured on an immobilised array of probes which are arranged at defined locations to act as bait for specific cells or fragments of cells. The binding of cells or fragments of cells can then be quantified by examination using the interferometer apparatus and/or interferometry methods described herein.

Digital nucleic acid testing Following the creation of an emulation in which nucleic acids are associated with beads which have specific nucleic acids attached to their surface, the nucleic acids are amplified using DNA amplification enzymes (requiring either thermal cycling or isothermal amplification). The resultant increase in mass on the surface of the bead can be identified using the interferometer apparatus and/or interferometry methods described herein. The bead size and composition can also vary to enable the identification of multiple different nucleic acid species from the same sample.

Claims

Claims 1. Optical apparatus for measuring characteristics of a measurement object, the apparatus comprising an illumination portion and, detection portion and a processing portion: the illumination portion comprising: means for producing at least one pair of spatially separated areas of illumination for illuminating a surface of said measurement object to produce an associated light field from which said characteristics of said measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: a component corresponding to interference between said areas of illumination; wherein said means for producing at least one pair of spatially separated areas of illumination is operable to: illuminate a measurement site on the measurement object with at least one of said spatially separated areas of illumination; and illuminate a reference site on the measurement object with at least one other of said spatially separated areas of illumination; the detection portion comprising: means for detecting light and for outputting signals dependent on the intensity of the detected light; means for receiving said light field from the measurement object resulting from said illumination of the measurement object with the at least one pair of said spatially separated areas of illumination, the light field resulting from each pair containing said component corresponding to interference between said areas of illumination; means for directing the received light field onto the light detecting means; the processing portion comprising: means for analysing said signals output by said detecting means to measure said characteristics of said measurement object.
2. Optical apparatus as claimed in claim 1 wherein said means for producing the at least one pair of spatially separated areas of illumination comprise shearing optics for shearing an incoming beam of light into at least two sheared beams of mutually coherent light, each sheared beam representing a respective source of one of said spatially separated areas of illumination.
3. Optical apparatus as claimed in claim 2 further comprising optics for transforming said at least two sheared beams into at least two parallel beams each parallel beam representing a respective source of one of said spatially separated areas of illumination.
4. Optical apparatus as claimed in any of claims 1 to 3 wherein said light field comprises a plurality components (e.g. in the form of diffraction fringes) having an increased power at spatial frequencies corresponding to said interference between said areas of illumination.
5. Optical apparatus as claimed in claim 4 wherein said analysing means is operable to analyse said signals output by said detecting means to determine changes in said components having an increased power and to measure a difference between a first phase of one of said at least one of said areas of illumination and a second phase for the other of said areas of illumination based on said determined changes in said components having an increased power.
6. Optical apparatus as claimed in any of claims 1 to S wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics of said surface of said measurement object associated with an effective difference between an optical path length for at least one of said areas of illumination and an optical path length for another of said areas of illumination.
7. Optical apparatus as claimed in claim 6 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics comprising a rotation of said surface of said measurement object to cause said effective difference between an optical path length for at least one of said areas of illumination and an optical path length for the other of said areas of illumination.
8. Optical apparatus as claimed in any of claims 1 to 7 wherein said means for producing spatially separated areas of illumination is operable to illuminate a measurement object with at least three spatially separated areas of illumination, wherein said at least three spatially separated areas of illumination are arranged to allow measurement for the measurement object to be performed for each of at least two axis of rotation.
9. Optical apparatus as claimed in claim 8 wherein said detection portion comprises means for spatially filtering said light field associated with said at least three spatially separated areas of illumination to produce a light field associated with two of said separated areas of illumination whereby to select an axis of rotation for which measurement is to be performed.
10. Optical apparatus as claimed in claim 9 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics comprising a rotation of a surface of said measurement object about said selected axis.
11. Optical apparatus as claimed in any of claims 1 to 10 wherein said detecting means comprises a point detector.
12. Optical apparatus as claimed in claim 11 further comprising means for modulating phase of at least one of said spatially separated areas of illumination, using a known phase modulation, whereby to allow said analysing means to determine differences in phase associated with characteristics of said measurement object by analysing phased with reference to said known phase modulation.
13. Optical apparatus as claimed in any of claims 1 to 10 wherein said detecting means comprises a one dimensional detector (e.g. a linear detector or linear array detector).
14. Optical apparatus as claimed in any of claims 1 to 10 wherein said detecting means comprises a two dimensional detector.
15. Optical apparatus as claimed in any of claims ito 14 wherein said means for producing at least one pair of spatially separated areas of illumination is operable to provide said spatially separated areas of illumination as two spots of illumination on a surface of a measurement object.
16. Optical apparatus as claimed in any of claims ito 14 wherein said means for producing at least one pair of spatially separated areas of illumination is operable to provide said spatially separated areas of illumination as two lines of illumination.
17. Optical apparatus as claimed in claim 16 wherein said analysing means is operable to analyse respective signals output by said detecting means for each of a plurality of different parts of said lines of illumination, whereby to measure characteristics of said measurement object at a plurality of different locations, each location being associated with a different respective part of said lines of illumination.
18. Optical apparatus as claimed in any of claims ito 17 wherein said means for producing at least one pair of spatially separated areas of illumination comprises means for scanning the spatially separated areas of illumination across a measurement object (e.g. without moving the apparatus from one location to another).
19. Optical apparatus as claimed in claim 18 wherein said scanning means comprises at least one mirror.
20. Optical apparatus as claimed in claim 18 or 19 wherein said scanning means comprises at least one scanning lens (e.g. an F over theta lens).
21. Optical apparatus as claimed in any of claims 1 to 20 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics of said measurement object associated with an effective difference between: an optical path length for the at least one area of illumination illuminating said measurement site; and an optical path length for the at least one other area of illumination illuminating said reference site.
22. Optical apparatus as claimed in any of claims 1 to 21 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics, of said measurement object, associated with molecular surface binding at the measurement site.
23. Optical apparatus as claimed in claim 22 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics, of said measurement object, associated with the occurrence of binding events associated with a change in optical path length.
24. Optical apparatus as claimed in claim 23 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics, of said measurement object, associated with the occurrence of binding events associated with an increase in optical path length.
25. Optical apparatus as claimed in claim 23 wherein said analysing means is operable to analyse said signals output by said detecting means to measure characteristics, of said measurement object, associated with the occurrence of binding events associated with a decrease in optical path length.
26. Optical apparatus as claimed in any of claims 22 to 24 wherein said means for producing at least one pair of spatially separated areas of illumination is operable to illuminate at least two further reference sites on the measurement object with at least one further pair of spatially separated areas of illumination; wherein said analysing means is operable to analyse said signals output by said detecting means for illumination incident on said at least two further reference sites to measure characteristics, of said measurement object, associated with rotation of said measurement object; and wherein said analysing means is operable to use said measured characteristics associated with rotation of said measurement object to mitigate the effect of said rotation said measures characteristics associated with molecular surface binding.
27. Optical apparatus as claimed in any of claims 1 to 24 further arranged for inducing surface plasmon resonance while performing said measurement.
28. Optical apparatus as claimed in any of claims 1 to 27 wherein said measurement object is located in an optically transparent medium (e.g. a medium having a refractive index greater than or equal to 1) and said illumination and detection portions are provided on either side of said optically transparent medium.
29. Optical apparatus as claimed in any of claims 1 to 27 wherein said measurement object is located in an optically transparent medium (e.g. a medium having a refractive index greater than or equal to 1) and said illumination and detection portions are provided on the same side of said optically transparent medium.
30. Optical apparatus as claimed in claim 28 or 29 wherein said measurement object is optically transparent having a refractive index that is different to said refractive index of said transparent medium.
31. Optical apparatus as claimed in claim 30 wherein said analysing means is operable to measure characteristics of said measurement object based on differences in phase associated with differences in said refractive indexes.
32. Optical apparatus as claimed in any of claims 28 to 31 wherein said analysing means is operable to measure characteristics of a measurement object comprising a particle flowing in said transparent medium, past said areas of illumination, the characteristics comprising a size of said particle.
33. Illumination apparatus for use as said illumination portion of the optical apparatus of any of claims 1 to 32, the illumination apparatus comprising: said means for producing at least one pair of spatially separated areas of illumination for use in measuring said characteristics of said measurement object, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path.
34. Detection apparatus for use as said detection portion, of the optical apparatus of claims 1 to 32, the detection apparatus comprising: said means for detecting light and for outputting a signal dependent on the intensity of the detected light; said means for receiving a light field from the measurement object resulting from illumination of the measurement object with at least one of said spatially separated areas of illumination; and said means for directing the received light field onto the light detecting means.
35. Signal processing apparatus for use as said processing portion, of the optical apparatus of claims 1 to 32, the signal processing apparatus comprising said means for analysing said signals output by said detecting means to measure said characteristics of said measurement object.
36. A method performed by optical apparatus for measuring characteristics of a measurement object, the apparatus comprising an illumination portion and, detection portion and a processing portion, the method comprising: the illumination portion: producing at least one pair of spatially separated areas of illumination for illuminating a surface of said measurement object to produce an associated light field from which said characteristics of said measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: a component corresponding to interference between said areas of illumination; wherein said at least one pair of spatially separated areas of illumination: illuminates a measurement site on the measurement object with at least one of said spatially separated areas of illumination; and illuminates a reference site on the measurement object with at least one other of said spatially separated areas of illumination; the detection portion: receiving said light field from the measurement object resulting from said illumination of the measurement object with the at least one pair of said spatially separated areas of illumination, the light field resulting from each pair containing at least said component corresponding to interference between said areas of illumination; directing the received light field onto light detecting means; detecting light at the detecting means and outputting signals dependent on the intensity of the detected light; the processing portion: analysing said signals output by said detecting means to measure said characteristics of said measurement object.
37. A method performed by illumination apparatus, the method comprising: producing at least one pair of spatially separated areas of illumination for illuminating a surface of a measurement object to produce an associated light field from which said characteristics of said measurement object can be measured, wherein the areas of illumination are mutually coherent and are each provided via a substantially common path such that the light field produced by the illumination of the surface of the measurement object comprises: a component corresponding to interference between said areas of illumination; wherein said means for producing at least one pair of spatially separated areas of illumination is operable to: illuminate a measurement site on the measurement object with at least one of said spatially separated areas of illumination; and illuminate a reference site on the measurement object with at least one other of said spatially separated areas of illumination.
38. A method performed by detection apparatus for detecting a light field produced using the method of claim 37, the method performed by the detection apparatus comprising: receiving said light field from the measurement object resulting from said illumination of the measurement object with the at least one pair of said spatially separated areas of illumination, the light field resulting from each pair containing at least said component corresponding to interference between said areas of illumination.
39. A method performed by signal processing apparatus for processing signals output by as part of the method of claim 38, the method performed by signal processing apparatus comprising: analysing said signals output by said detecting means to measure said characteristics of said measurement object.