Abstract
A device configured to detect signals indicative of a binding event in an assay of interest is provided. The device comprising a beam manipulator configured to modify an incident beam to form a beam with a sharp-edged intensity cut-off; a test site having immobilised thereon at least one component of the assay of interest; wherein the device is configured so that the sharp-edged intensity cut-off beam produced by the beam manipulator is incident, in use, on the test site at such an angle to facilitate Total Internal Reflection (TIR); wherein the device is configured so that the sharp-edged intensity cut-off beam produced by the beam manipulator substantially conforms to the shape and size of the test site; a detector configured to receive a signal indicative of a binding event in the assay of interest; wherein the test site is located within a microfluidic channel.
Claims
1: A device configured to detect signals indicative of a binding event in an assay of interest; the device comprising: a beam manipulator configured to modify an incident beam to form a beam with a sharp-edged intensity cut-off; a test site having immobilised thereon at least one component of the assay of interest; wherein the device is configured so that the sharp-edged intensity cut-off beam produced by the beam manipulator is incident, in use, on the test site at such an angle to facilitate Total Internal Reflection (TIR); wherein the device is configured so that the sharp-edged intensity cut-off beam produced by the beam manipulator substantially conforms to the shape and size of the test site; and a detector configured to receive a signal indicative of a binding event in the assay of interest; wherein the test site is located within a microfluidic channel.
2: The device according to claim 1, wherein the beam manipulator comprises an optical element with an aperture, wherein the beam manipulator is configured to modify the amplitude of the incident beam to form a beam with a sharp-edged intensity cut-off at the test site.
3. (canceled)
4: The device according to claim 2, wherein the aperture is a hard aperture.
5: The device according to claim 2, wherein the aperture is a soft aperture.
6: The device according to claim 1, wherein the beam manipulator is configured to modify the phase of the incident beam to form a beam with a sharp-edged intensity cut-off at the test site.
7: The device according to claim 6, wherein the beam manipulator comprises one or more refractive optical elements.
8: The device according to claim 6, wherein the beam manipulator comprises one or more reflective optical elements.
9: The device according to claim 6, wherein the beam manipulator comprises one or more diffractive optical elements.
10: The device according to claim 1, wherein the beam manipulator comprises an optical element configured to transform the incident beam into an Airy disc and a focusing lens or mirror configured to produce a sharp-edged intensity cut off beam at the test site.
11: The device according to claim 10, wherein the beam manipulator further comprises a phase element.
12: The device according to claim 1, wherein the beam manipulator further comprises a multimode optical waveguide and a lens adjacent to the output face of the waveguide.
13: The device according to claim 1, wherein the beam manipulator is further configured to modify the incident beam to form a beam with a reduced speckle pattern; and wherein the device is configured so that the beam with the reduced speckle pattern produced by the beam manipulator is incident, in use, on the test site at such an angle to facilitate Total Internal Reflection (TIR).
14: The device according to claim 13, wherein the reduced speckle pattern is created using a variable phase adjustment that varies across the beam.
15: The device according to claim 1, wherein the beam manipulator comprises a vibrating plate.
16: The device according to claim 1, wherein the beam manipulator comprises a dynamic mode scrambler.
17: The device according to claim 1, wherein the beam manipulator comprises a rotating diffuser.
18: The device according to claim 1, wherein the beam manipulator further comprises a multimode optical waveguide and a lens to couple the light into the multimode waveguide.
19: The device according to claim 6, wherein the lens is continuously perturbed to form a beam with a reduced speckle pattern.
20: The device according to claim 1, wherein the beam manipulator further comprises a diffuser plate which is continuously perturbed to form a beam with a reduced speckle pattern.
21-23. (canceled)
24: The device according to claim 1, wherein the test site has a rectilinear geometry.
25-26. (canceled)
Description
FIGURES
[0090] The present invention will now be described, by way of example only, with reference to the accompanying figures in which:
[0091] FIG. 1 shows, schematically, a simple TIR microscope architecture;
[0092] FIG. 2 shows, schematically, a TIR illumination region;
[0093] FIGS. 3A to 3D show various illumination scenarios suitable for achieving TIR;
[0094] FIG. 4A shows a Gaussian beam profile with relevant dimensions labelled;
[0095] FIG. 4B shows a beam profile for an ideal Gaussian beam with n=2;
[0096] FIG. 4C shows a beam profile for an ideal super-Gaussian beam with n=10;
[0097] FIG. 4D shows a beam profile of the measured output after conditioning with a square core fibre and an ideal Gaussian beam profile;
[0098] FIG. 5A illustrates beam shaping as a Gaussian input beam is incident on a hard aperture;
[0099] FIG. 5B illustrates beam shaping as a Gaussian input beam is incident on a soft aperture;
[0100] FIG. 6 shows a refractive or reflective phase element shaping a collimated Gaussian input beam into a Flat-Top intensity distribution after a certain propagation distance;
[0101] FIG. 7 shows a second refractive or reflective phase element modifying the phase of the diverging Flat-Top intensity distribution of FIG. 6 to generate a flat wavefront;
[0102] FIG. 8 shows a diffractive element converting a collimated Gaussian input beam to a Flat-Top, sharp-edged intensity cut off distribution, and a second diffractive element used to generate a collimated beam;
[0103] FIG. 9 shows an optical setup used to convert a Gaussian input beam into an Airy Disc, and subsequently to produce a flat wavefront and a collimated beam;
[0104] FIG. 10A shows a cross section of a multimode waveguide with a square core;
[0105] FIG. 10B shows a cross section of a multimode waveguide with a rectangular core;
[0106] FIG. 10C shows a cross section of a multimode waveguide with a circular core;
[0107] FIGS. 11A and 11B show a beam profile and corresponding central line profile for a circular core cross section fibre;
[0108] FIGS. 11C and 11D show a beam profile and corresponding central line profile for a square core cross section fibre;
[0109] FIGS. 11E and 11F show a beam profile and corresponding central line profile for a rectangular core cross section fibre;
[0110] FIG. 12 illustrates the conversion of a Gaussian input beam to a flat-top, sharp-edged intensity cut-off beam at the image plane of a lens using a square-core optical fibre;
[0111] FIGS. 13A and 13B show illumination region images and corresponding intensity profiles achieved with laser illumination;
[0112] FIGS. 13C and 13D show illumination region images and corresponding intensity profiles achieved by optimising the illumination beam according to the present invention and utilising an LED light source;
[0113] FIGS. 14A and 14B show a TIR image of a bare auto-fluorescent substrate and an intensity profile corresponding to the cross-section;
[0114] FIG. 14C shows a fluorescent micro-array on the substrate of FIG. 14A, captured using the sharp-edged intensity cut-off beam shown in FIG. 14C;
[0115] FIG. 15A shows the intensity distribution at the output face of a square-core optical fibre when laser radiation is coupled to the fibre;
[0116] FIG. 15B shows the intensity profile through the centre of the fibre of FIG. 15A;
[0117] FIG. 16A shows the intensity distribution at the output face of a square-core optical fibre when laser radiation is coupled to the fibre and the fibre is attached to a vibrating plate;
[0118] FIG. 16B shows the intensity profile through the centre of the fibre of FIG. 16A;
[0119] FIGS. 17A and 17B show a square core fibre imaged without and with speckle reduction, respectively and
[0120] FIGS. 18A and 18B show TIR images of a fluorescent micro-array without and with speckle reduction respectively.
DETAILED DESCRIPTION OF FIGURES
[0121] Referring to FIG. 1, a simple TIR microscope architecture is shown, comprising an illumination source 2, an input beam 4, a higher refractive index medium 6, a lower refractive index medium 8, an output beam 12, imaging optics 14 and an image sensor 16.
[0122] TIR occurs at the interface between the higher refractive index material 6 and the lower refractive index material 8, as the incident light above a critical angle impinges at the interface, resulting in a TIR region 18.
[0123] At the TIR region 18, an evanescent, exponentially decaying light field is established in the lower refractive index medium 8. This evanescent field is restricted to the region immediately adjacent to the interface, and in typical microscopy scenarios where the higher refractive index medium 6 is glass and the lower refractive index medium 8 is a liquid sample, the penetration depth is on the order of 100 nm.
[0124] FIG. 2 shows a schematic of the test site of the present invention. The test site is also the location of the TIR region 18. Capture components of an assay of interest 24 are immobilised at the interface of the higher refractive index material 6 and the lower refractive index material 8. The capture components 24 are therefore immobilised within the evanescent field illumination region 26 and can be utilised for the detection of a target analyte 22 in a liquid sample. In the example shown in FIG. 2, the lower refractive index medium 8 is a liquid sample containing a target analyte 22 bound to a label 20. The target analyte 22 may be a specific protein in a biological liquid sample, and the label 20 may be a fluorescent or highly scattering particle such as a nanoparticle. The capture components 24 could be antibodies specific to the target protein. The label 20 bound to the target analyte 22 allows for the detection of the target analyte 22 by producing a measurable signal. The selected illumination achieved by the limited penetration depth of the evanescent field can be extremely advantageous, since the imaging system can gather signal efficiently from the evanescent field illumination region 26, whilst non-illuminated regions 28 of the lower refractive index medium 8, do not contribute to the image. Target analyte 22 located outside of the evanescent field illumination region 26 does not interfere with the image, thus achieving a high signal to background ratio. An imaging system consisting of a series of lenses or imaging optics 14 can be used to image the evanescent field illumination region 26 onto an image sensor 16.
[0125] The device of the present invention optimises a beam of light for the improved detection of signals indicative of a binding event in an assay using TIR microscopy. The beam of light must satisfy the requirements for TIR at the TIR region 18. FIG. 3 shows various illumination scenarios which could achieve TIR. The incident radiation must be incident on the TIR region 18 at an angle larger than the critical angle, whereby the critical angle, .sub.c, is defined by:
[00003]
where n.sub.2 is the refractive index of the lower refractive index medium 8 which can be the sample material, and n.sub.1 is the refractive index of the higher refractive index medium 6 that the illumination beam travels through. For example, if the sample material is water, with refractive index n.sub.2=1.33, and the refractive index of the higher refractive index medium 6 is borosilicate glass, with refractive index n.sub.1=1.47, then .sub.c=64.8. In this example, the illumination would need to be incident on the TIR region 18 at angles greater than 64.8, which acts as a lower limit for the incident angle. An upper limit is naturally defined by 90, since at this angle the light is travelling along the surface. Therefore, the range of angles that can exist in the illumination beam, .sub.BEAM, must satisfy:
[00004]
[0126] The range on angular tolerances depends on the geometry of the higher refractive index 6, where the beam is incident on the higher refractive index medium 6, the beam width, the desired geometry at the TIR region 18 and where the TIR region 18 is located. FIG. 3 illustrates the substantial effect these can have on the TIR region 18. FIG. 3A shows the properties of the incident beam at the TIR region 18 with an input beam 4 with parallel rays. FIGS. 3B, 3C and 3D show the properties of the incident beam at the TIR region 18 with an input beam 4 with converging rays. FIGS. 3B, 3C and 3D also show the beam properties at the TIR region 18 with varying location of the TIR region 18, and with varying input beam 4 width.
[0127] The key requirement of the present invention is that the beam manipulator forms a beam with a sharp-edged intensity cut-off the test site, which is incident, in use, at the test site at such an angle to facilitate TIR. The beam should have a significantly sharper intensity cut-off than a Gaussian laser beam. This is important, since this property allows the desired regions of the sample to be illuminated whilst minimising the illumination of other regions. The sharpness parameter, S, defines the sharpness of the intensity cut-off. It is calculated by considering the ratio of the full-width half-maximum (FWHM) of the beam to the distance between the 10% and 90% intensity points of the beam. Ideally, this ratio would be as large as possible to maximise the sharpness of intensity cut-off. To be effective in this context, the S value for the shaped beam must be at least two times greater than the S value for the Gaussian beam. The intensity profile of a Gaussian beam is described by:
[00005]
where l.sub.0 is the on-axis intensity, r is the radial position, w is the beam width, defined as the radial position where the intensity has reduced to exp(2) or approximately 13.5% of l.sub.0, and n is the order, which is equal to 2 for a fundamental Gaussian laser beam. The above equation can be used to calculate the radial position for an arbitrary fraction of the on-axis intensity, X, given by:
[00006]
[0128] Using this equation and an assumed beam width of w=250 m, the radial position of the 10% intensity point (X=0.1) is given by 268.2 m, and the radial position of the 90% intensity point (X=0.9) is given by 57.4 m, thus the distance between these points is 210.8 m. The radial position of the half-maximum point (X=0.5) is given by 147.2 m, and so the FWHM is 294.4 m. Finally, the ratio S for this beam, and any beam conforming to a fundamental Gaussian, is 1.40.
[0129] FIG. 4A shows the distance between 10% and 90% of the on-axis intensity, and the FWHM for the example of a Gaussian beam with a 250 m beam width. The beam profile for an ideal Gaussian beam with n=2 is shown in FIG. 4B.
[0130] If this analysis is repeated for a super-Gaussian beam of order n=10, a higher value of S=6.68 is obtained. The beam profile for an ideal super-Gaussian beam with n=10 is shown in FIG. 4C. The present invention facilitates the generation of a beam that exhibits a sharp-edged intensity discontinuity at the perimeter of the beam profile, which exists in a collimated manner, and with a constant intensity across the beam cross-section. The device of the present invention comprises a beam manipulator, which manipulates the phase, amplitude, or both the phase and amplitude of a beam, to produce a Flat-Top, sharp-edged beam as is also shown in FIG. 4D. A value for S of approx. 4.09 was determined from the measured output after conditioning with a square core fibre shown in FIG. 10A.
[0131] In a preferred embodiment, the test site may have a rectilinear geometry. By shaping the beam such that it substantially conforms to the shape and size of the test-site, interactions between the beam and test site structures can be minimised. The shaped beam can interrogate a maximised space at the test site and improve the overall image quality compared to interrogation with a Gaussian beam.
[0132] Referring to FIG. 5, the beam manipulator may be configured to modify the amplitude of a Gaussian incident beam 30. As shown in FIG. 5A, the beam manipulator may be an optical element with a hard aperture 32. The incident beam 30 is directed towards the optical element with the hard aperture 32, and any light that is not incident on the aperture opening 34 is absorbed or reflected. The light from the incident beam 30 that is incident on the aperture opening 34 is transmitted. A shaped output beam 36 is produced.
[0133] At the position immediately after the aperture opening 34, the shaped output beam 36 exactly conforms to the shape of the aperture opening 34. The output beam 36 has a sharp-edged profile, with a flatness across the beam dictated by the flatness of the portion of the beam sampled by the aperture opening 34. As the output beam 36 propagates from the aperture opening 34 in the direction shown by the arrow 38 in FIG. 5A, the effects of diffraction increase the complexity of the beam profile, and the aperture shape and sharp-edged nature of the beam is reduced as the distance from the aperture increases. The distance over which the beam displays an acceptable spatial profile is at least partially dictated by the quality of the input beam 30, the wavelength of the input beam 30, the size of the input beam 30 the divergence of the input beam 30, the size of the aperture opening 34, and the level of acceptability defined for the specific application. The aperture can be imaged with a suitable magnification, to further control the size, position, divergence, and propagation of the shaped output beam 36.
[0134] Referring to FIG. 5B, the beam manipulator may be an optical element with a soft aperture 40. The Gaussian incident beam 30 is directed towards the soft aperture 40. The soft aperture 40 has a transmission that varies spatially across the aperture. The shape of the output beam 42 immediately after the soft aperture 40 is determined by the input beam profile and the transmission profile of the soft aperture 40. Compared to the output beam 36 obtained using a hard aperture 32, the output beam 42 obtained with the soft aperture 40 is not as sharp-edged. As the output beam 42 propagates from the soft aperture 40 in the direction shown by the arrow 44 in FIG. 5B, the effects of diffraction increase the complexity of the beam profile. The output beam produced by the soft aperture 40 has less severe effects of diffraction with propagation length compared to the beam produced with the hard aperture 32. The aperture can be imaged with a suitable magnification, to further control the size, position, divergence, and propagation of the shaped output beam.
[0135] The beam manipulator may be configured to modify the phase of an input beam using a refractive, reflective or diffractive optical element to form a beam with a sharp-edged intensity cut-off.
[0136] Referring to FIG. 6, a collimated Gaussian input beam 46 with a known intensity distribution can be directed towards a refractive or reflective phase element 48 to convert the input beam 46 to a Flat-Top intensity distribution 50 after a certain propagation distance. The refractive or reflective phase element 48 may be a specifically designed lens or mirror. The distribution of intensity, indicated by the spacing of the rays 52, can be redistributed by the refractive or reflective phase element 48, which is designed to appropriately modify the wavefront, by imparting a specific position dependent phase change to the beam.
[0137] Referring to FIG. 7, a second refractive or reflective phase element 54 can be placed at the position of the Flat-Top diverging output beam 50 shown in FIG. 6 to selectively change the phase of the diverging Flat-Top intensity distribution to generate a flat wavefront and a shaped collimated output beam 56. The desired sharp-edged intensity distribution is maintained. The second phase element 54 may be a specifically designed lens or mirror. The beam will naturally diffract with propagation length from the phase element 48, but the inclusion of a second refractive or reflective phase element 54 can significantly increase the distance over which the beam retains the desired properties.
[0138] Referring to FIG. 8, the beam manipulator may comprise one or more diffractive optical elements 58 and 60. The diffractive optical elements 58 and 60 may be transmissive or reflective. The diffractive optical elements 58 and 60 may be diffractive diffusers or diffractive beam shapers. The diffractive optical element 58 modifies the phase of a collimated Gaussian input beam 46 to a Flat-Top sharp-edged intensity beam. A second diffractive element 60 located at the target plane modifies the phase of the beam to generate flat wavefronts and thus a shaped collimated output beam 62.
[0139] The beam manipulator may comprise an optical element configured to transform the incident beam into an Airy disc. Referring to FIG. 9, an Airy disc conversion optic 64 converts the Gaussian incident beam 30 into an Airy disc and is subsequently focussed by a lens or mirror 66. The Airy disc conversion optic 64 manipulates the amplitude, or phase, or both the amplitude and the phase of the beam. The focussing lens or mirror 66 forms the Fourier transform of the Airy disc at the focal plane, to produce a Flat-Top beam. A phase element 68 is positioned at the focal plane to modify the phase of the beam to produce flat wavefronts and a shaped collimated output beam 70. The phase element 68 is positioned one focal length from the focussing lens or mirror 66.
[0140] The beam manipulator may comprise an optical waveguide configured to modify an incident beam with a combination of both amplitude and phase control to form a beam with a sharp-edged intensity cut off. The optical waveguide may be a multimode optical waveguide such as a multimode fibre 72, or may be a light pipe, or any other suitable device.
[0141] As shown in FIG. 10, the multimode fibre 72 comprises a fibre cladding 74. The cross-section of the guided light 78 will conform to that of the core 76 of the waveguide 72. Referring to FIGS. 10A and 10B, preferably, the core 76 may have a rectilinear geometry. Alternatively, the core 76 may have a circular geometry as shown in FIG. 10C. By utilising a waveguide with a core 76 that has a square or rectangular cross-section, guided light with low temporal coherence, uniform intensity profile and a square or rectangular cross-section can be created. The core 76 of the multimodal fibre 72 can be shaped to conform to the geometry of the test site within the evanescent field illumination region 26 and the image sensor 16.
[0142] FIG. 11 depicts the beam profiles and corresponding central line profiles achieved with the various core fibre cross-section geometries shown in FIGS. 10A to 10C. The beam profiles are taken at the image plane. FIGS. 11A and 11B show the beam profile and corresponding central line profile for a circular core cross-section waveguide. FIGS. 11C and 11D show the beam profile and corresponding central line profile for a square geometry core cross-section waveguide, and FIGS. 11E and 11F show the beam profile and corresponding central line profile for a rectangular geometry core cross-section waveguide. FIG. 11 shows the flat-top nature of the beam is improved for the square and rectangular geometry core cross sections compared to the circular core cross section beam profile. The rectilinear geometry core cross-sections have more efficient mode mixing. The shape of the illumination region can be adapted to conform to the field of view of an image sensor.
[0143] An example experimental setup used to produce the intensity profile shown in FIGS. 11C and 11D uses a single-colour red LED (M625L4, Thorlabs Inc.), butt-coupled to a 2 m length of square core multimode fibre with a core side length of 150 m (M101L02, Thorlabs, Inc.). The output of which was imaged using an 8 mm focal length aspheric lens (C240TMD-B, Thorlabs, Inc.), and the subsequent image was detected at the image plane with a CMOS image sensor (DCC1645C, Thorlabs Inc.).
[0144] Referring to FIG. 12, a Gaussian incident beam 30 is directed towards a multimode fibre 72. As shown in FIG. 12, the multimode fibre 72 may be a square-core fibre. The incident beam 30 may be coherent or incoherent light. The multimodal fibre 72 traps a portion of light within the core of the waveguide to create a guided light beam. If the waveguide is highly multimoded, the distribution of power in the core of the fibre 72 can be uniformly spread across the core cross-section, creating a uniform intensity profile. This is especially advantageous when coupled with an illumination source of low spatial coherence such as LED emission, which emits light over a wide range of angles with low directionality.
[0145] Excitation of higher-order modes with the fibre 72, and mode-mixing between these modes leads to a Flat-Top intensity profile beam 80 at the output face of the fibre. The beam at the output of the fibre 80 is sharp-edged with a shape that conforms to the square shape of the fibre 72. Once the light exits the output face of the multimodal waveguide 72 it will diffract, and consequently loses the properties of uniform intensity profile and square or rectangular cross-section. Therefore, an imaging lens 82 magnifies the output of the fibre 80, to produce a Flat-Top sharp-edged beam 84 at an image plane 86. This allows the formation of a beam of uniform intensity profile and square or rectangular cross-section at a specific distance. The Flat-Top sharp-edged beam 84 has larger dimensions than the fibre 72 and a lower divergence than the beam exiting the fibre 80. The focal length and position of the imaging lens 82 determines the dimensions, position, depth-of-focus, and range of angles in the beam.
[0146] In order to achieve a narrow angular range the imaging lens 82 can be positioned slightly further than focal length of the imaging lens 82, producing a magnified image. Careful consideration of the waveguide and lens design and lens position can achieve a beam with an appropriate depth-of-focus and dimensions at a suitable distance i.e., at the test site.
[0147] FIG. 13 shows TIR images and intensity profiles from five dumbbell-shaped spots of surface-bound antibody. In this example, the sample consisted of a liquid solution which was brought into contact with a glass substrate. The sample contained a specific protein which was the target analyte. The protein could bind to the antibodies surface-bound to the glass surface, and to free antibodies in solution which were labelled with gold nanoparticles. TIR occurred at the glass/sample interface. A measurable signal was obtained when double-recognition events occurred, i.e., the protein bound both with a surface-bound antibody and a labelled mobile antibody.
[0148] FIGS. 13A and 13B show TIR images and intensity profiles obtained using a laser illumination source without beam shaping and a simple TIR imaging set up (as shown in FIG. 1). FIG. 13B shows a vertical intensity line profile through the right-hand lobe of the spots attained through laser illumination.
[0149] FIG. 13C shows the images obtained from the five dumbbell-shaped spots of surface-bound antibody using the illumination architecture shown in FIG. 12, utilising an LED source coupled to a square-core fibre to produce a rectangular sharp-edged intensity cut-off at the test site. FIG. 13D shows a vertical intensity line profile through the right-hand lobe of the spots. The comparison between FIGS. 13A and 13B, and FIGS. 13C and 13D, clearly shows a more uniform intensity profile and significantly more even illumination distribution across the five spots attained using the illumination architecture of the present invention.
[0150] FIG. 14 shows TIR images of a bare auto-fluorescent substrate with a cross-section plot and a fluorescent micro-array on a substrate with low auto-fluorescence. FIG. 14A shows the auto-fluorescence from the excitation beam profile with hard-edged shaped beam. The bright vertical band in the image corresponds to the area of the substrate exposed with the beam and the dark vertical bands on the edges of the image correspond to the image background where the beam intensity is negligible. An auto-fluorescent substrate is used in this example to capture the beam profile without removing fluorescence imaging filters. The horizontal dashed line represents the location of the intensity cross-section plotted in FIG. 14B. The intensity plotted is a box average of 10 nearest vertical pixels for each horizontal pixel. FIG. 14C shows an image of a micro-array captured using the hard-edged shaped excitation beam. The micro-array consists of a 9 by 3 array of fluorescent spots. The spots are fluorescently labelled surface-bound antibodies. The micro-array is imaged on a higher quality substrate with lower auto-fluorescence to enhance the contrast between the spots and the image background. The scale bars shown in FIGS. 14A and 14C are 500 m.
[0151] In some embodiments, in which the input light is coherent light such as laser radiation, a granular speckle pattern can be created across the beam profile, resulting from the interference of the different supported modes. Referring to FIG. 15A, the intensity distribution at the output face of the square core fibre 72 of FIG. 12 is shown. Referring to FIG. 15B, the intensity profile through the centre of the fibre 72 is shown. The grainy nature of the image is typical of the speckle pattern.
[0152] To circumvent the issue of speckle, the fibre 72 shown in FIG. 12 can be continuously perturbed. For example, the fibre 72 may be placed on a vibrating plate. The various modes excited and/or various mode coupling that exists is continuously varied and a large number of different speckle patterns are produced over a particular time period. When averaged over this time period, the speckle patterns can act to cancel each other out to produce an averaged intensity profile which is significantly smoother. Referring to FIG. 16A, an image of the same fibre 72 shown in FIG. 15 is shown when the fibre 72 is attached to a vibrating plate and the exposure time of the image sensor is set to one second. The effect of speckle can be seen to be minimised. Referring to FIG. 16B, the intensity profile through the centre of the fibre 72 is significantly smoother. This effect is largely avoided using incoherent light such as LEDs, which exhibits speckle patterns with different minima and maxima for the different frequency components, which can act to cancel out the granularity to produce a smooth and homogeneous intensity distribution.
[0153] FIGS. 17A and 17B show a square core fibre 72 without and with speckle reduction, respectively.
[0154] In relation to quantifying an acceptable level of speckle reduction, the degree of speckle can be quantified by illuminating an image sensor with the beam of interest and determining the relative pixel intensity variations across the image. A commonly used technique for quantifying speckle in the literature is the Speckle Contrast, C, defined as:
[00007]
where I is the pixel intensity value, <I> is the mean pixel intensity and .sub.I is the standard deviation of the intensity values.
[0155] A region of interest of 151151 pixels is defined in both FIGS. 17A and 17B, by the box. The Speckle Contrast for the region of interest without speckle reduction is C=0.658, and with speckle reduction is C=0.087. Visually, inspecting FIGS. 17A and 17B there is a clear qualitative reduction in speckle and the calculation of C in each case shows a corresponding reduction in value.
[0156] The Speckle Contrast value is dependent on many factors including but not limited to: the beam size at the image sensor, background signal, the image sensor pixel size, the image sensor bit-depth and the image sensor noise properties. Specifying a particular value for C is non-trivial. However, specifying a value for C is appropriate. A value of C<0.2 may be an appropriate target requirement.
[0157] FIG. 18 shows TIR images of a fluorescent micro-array with and without speckle reduction. The micro-array consists of a 9 by 3 array of fluorescent spots. The spots are fluorescently labelled surface-bound antibodies. In this example, speckle reduction was achieved using a speaker driver as the speckle reducing device and the excitation beam source was a laser coupled to a square-core multimode optical fibre 72. The size of the excitation beam profile was matched to the size of the micro-array so that all spots in the micro-array were exposed simultaneously. The speaker driver was fixed to the optical fibre 72 such that the driver vibrations were coupled to the fibre 72 at a single point. The fibre 72 was oscillated using a square waveform at 184 Hz to reduce the observed speckle pattern on the array spots from the excitation beam.
[0158] FIG. 18A shows an image of the micro-array with the speckle reduction device turned OFF and FIG. 18B shows an image of the micro-array with the speckle reduction device ON. As shown in FIG. 18A, when the speckle reduction device is OFF, vertical bright and dark streaks are observed on the micro-array spots. The streaks are caused by the elongation of speckle pattern at the high angle-of-incidence required for TIR imaging. The scale bars in FIGS. 18A and 18B are 500 m.
[0159] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
[0160] and/or where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example A and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
[0161] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
[0162] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.
