Patent Publication Number: US-2022214259-A1

Title: Evanescent field resonance imaging microscopy apparatus and method

Description:
FIELD OF THE INVENTION 
     The invention generally relates to imaging and/or characterising the geometry of samples (e.g. particles, structures, substrates) using evanescent field scattering/resonance. 
     BACKGROUND TO THE INVENTION 
     Particle characterisation (e.g. determination of one or more size and/or shape parameters) is critical to many industrial processes for tuning product fabrication, formulation and quality control. It is routinely carried out, for example as part of an R&amp;D programme or as an element of quality control. It is routinely used in the formulation and manufacture of products in industrial sectors including pharmaceuticals, chemical processing, mining and minerals process, food and beverage, consumer products and coatings (e.g. paint) and many others. 
     There are many existing methods for particle characterisation. These can be roughly arranged into categories including microscopy methods (optical or otherwise), flow methods, diffraction methods and diffusion-based methods. Most of these methods assume spherically shaped particles, and when this is not the case, often require either ultrahigh-vacuum sample preparation or complicated chemical labelling. 
     The accuracy of standard optical microscopy using white light for bright-field image analysis techniques (e.g. extracting size and/or shape parameters of a particle from a video microscope image) is fundamentally limited by the wavelength of visible light, where in practice particle sizes and shapes are difficult or resolve below 1 micron. Observations can be made in liquids or in air where the particles must be fixed to a slide or substrate. Techniques such as scanning electron microscopy (SEM) or transmission electron microscopy (TEM) employ a beam of electrons in a way that is analogous to the way an optical microscope uses a beam of light to perform imaging. SEM/TEM techniques generally require extensive sample preparation and operate under vacuum. As SEM and TEM both use a beam of electrons, most preparations require either metal samples or metal coatings on the sample to prevent charring of the sample. Another sub-category of microscopy includes methods employing fluorescence. In these methods, the particles must be coated or impregnated with a fluorescent dye. Specific wavelengths of light are used to excite the fluorescence of these dyes (or molecules) such that only known spectrum of light is emitted. These techniques have found very successful and widespread applications in biology, but the challenge of chemically tagging or dyeing a particle involves an elaborate sample preparation and chemistry pathways. Additionally, obtaining the best possible spatial resolution using these techniques often requires the samples under observation to remain relatively static, further limiting industrial and commercial utility in time-sensitive or online applications. 
     Other techniques for particle sizing are generally based on flow (e.g. Taylor dispersion analysis, also called orifice plate methods), planewave light scattering/diffraction (e.g. dynamic light scattering or laser diffraction), or monitoring and quantifying the diffuse motion of particles in solution (e.g. nanoparticle tracking analysis, for example the Malvern NanoSight). These techniques usually explicitly assume spherical particles. 
     SUMMARY OF THE INVENTION 
     According to an aspect of the present invention, there is provided a method for characterising a sample located within an imaging region, the method comprising the steps of: generating one or more evanescent fields, each associated with a direction, within the imaging region; capturing an image of the imaging region; determining one or more sample characteristics of the sample according to a spatial intensity pattern resulting from an interaction between the, or each, evanescent field and the sample within the image. 
     In an embodiment, the one or more sample characteristics include one or more shape parameters. In an embodiment, the one or more sample characteristics include one or more size parameters. 
     In an embodiment, the intensity pattern comprises one or more local intensity maxima, and wherein the one or more sample characteristics are determined at least in part based on an identified location of the one or more local intensity maxima. The step of determining a size parameter and/or shape parameter of the sample includes determining a location of at least one surface of the sample based on at least one local light intensity maximum within the image. 
     Optionally, at least two evanescent fields are generated simultaneously and are each associated with a unique characterising spectrum. Also, or alternatively, according to an option, at least two evanescent fields are created according to a sequence, wherein the sequence includes at least one evanescent field generated after at least one other evanescent field. Each evanescent field is generated at a unique time such that no two evanescent fields are present within the imaging region at the same time. 
     The method may further comprise the steps of: identifying a plurality of local light intensity maxima associated with the evanescent fields; and determining sample size parameter(s) and/or shape parameter(s) consistent with the relative positions of the plurality of local maxima. Optionally, identifying a plurality of local maxima includes applying a filter for identifying a central location or locations of local maxima within the intensity pattern. A sample size parameter(s) and/or shape parameter(s) may be determined in dependence on the directions associated with each evanescent field. 
     Optionally, the image is captured by an image sensor coupled to an optical magnifier, such that the imaging region is viewable by the image sensor via the optical magnifier. 
     According to another aspect of the present invention, there is provided a sample characterising apparatus comprising an imaging sensor, an optical medium including a first surface above which a sample is positionable, and a plurality of light inputs each configured to direct light received by the light input into the optical medium from a unique direction such as to produce total internal reflection from the first surface when no sample is present, is wherein the imaging sensor is arranged to capture an image of a spatial intensity pattern due to a sample interacting with an evanescent field associated with each light input. 
     In an embodiment, at least one light input is controllable such that only one light input projects light into the optical medium at a time. Optionally, at least two light inputs are each associated with a unique characterising wavelength, and the imaging sensor is configured to image the first surface such that each light input is differentiable. 
     Each light input may be coupled to a light source. At least one light source may be a laser. At least one light source may be an LED light source—for example, the optical coupler may comprise the at least one LED light sources. 
     Optionally, for each light input, the angle at which light is projected into the imaging region is adjustable. 
     The apparatus may comprise a magnifier optically coupled to the imaging sensor, optionally wherein a magnification of the magnifier is adjustable. 
     According to yet another aspect of the present invention, there is provided a sample characterising system comprising the sample characterising apparatus of a previous aspect, the system configured to implement the method of a previous aspect. 
     As used herein, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In order that the invention may be more clearly understood, embodiments will now be described, by way of example, with reference to the accompanying drawing, in which: 
         FIG. 1  shows an evanescent field imaging apparatus according to an embodiment; 
         FIG. 2  shows a representation of total internal reflection; 
         FIG. 3A  shows an optical coupler having three coupling points; 
         FIG. 3B  shows an optical coupler having eight coupling points; 
         FIG. 3C  shows an embodiment wherein the light sources are components of the optical coupler; 
         FIG. 3D  shows an embodiment wherein the optical coupler includes a plurality of facets; 
         FIG. 4A  shows the operation of an imaging apparatus according to an embodiment without a sample present, wherein each light source is associated with a unique characterising wavelength; 
         FIG. 4B  shows the operation of an imaging apparatus according to an embodiment without a sample present, wherein each light source is associated with a unique temporal position; 
         FIG. 5A  shows an example of a sample being illuminated due to an interaction with the evanescent fields, according to an embodiment; 
         FIG. 5B  shows an example of a sample being illuminated due to an interaction with the evanescent fields, wherein the embodiment comprises a baffle region; 
         FIG. 6A  shows an example of a substantially spherical sample imaged by the imaging device of an embodiment; 
         FIG. 6B  shows an example of a sample with an irregular shape imaged by the imaging device of an embodiment; 
         FIG. 6C  shows an example of a sample showing characterising of a sample where the image highlights diffractive effects, according to an embodiment; 
         FIGS. 7 and 8  show example methods for operating the imaging device according to embodiments; 
         FIG. 9  shows an embodiment wherein the particles of the sample move across the imaging region; 
         FIG. 10  shows an embodiment utilising a single light source and a continuously variable light input direction; 
         FIG. 11  shows an example of imaging a sample; 
         FIG. 12  shows an example of imaging a sample comprising a plurality of particles; and 
         FIG. 13  shows another example of imaging a sample comprising particles of different size. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
       FIG. 1  shows an evanescent field imaging apparatus (imaging apparatus)  10  according to an embodiment. The imaging apparatus  10  comprises an optical coupler  14  configured to accept a sample  50 . The imaging apparatus  10  optionally also comprises a magnifier  12  comprising an arrangement of one or more lenses (not shown) optically coupled to an imaging sensor  13 . The magnifier  12  can be, for example, a compound microscope. The imaging sensor  13  can be, for example, a digital camera or a custom designed imaging sensor. The optical coupler  14  defines an imaging region  11  in which the sample  50  is locatable. The imaging apparatus  10  also comprises one or more light inputs  15 . The light inputs  15  are coupled to the optical coupler  14 , for example: using fibre optic cables  22 , via direction of one or more light beams (such as a laser beam(s)) through free air onto the light inputs  15 , or with directly coupled light sources  16  such as LEDs provided with the optical coupler  14 , to one or more light sources  16 . In  FIG. 1 , each light input  15  is uniquely coupled to a light source  16  such that there is a different light source  16  for each light input  15 . However, in other embodiments, one or more of the light inputs  15  are coupled to a common light source  16 —these embodiments will be described in more detail below. At least one light source  16  can be a laser. In another embodiment, at least one light source  16  is a LED source. It is anticipated that two or more different types of light source  16  can be utilised; for example, both LED sources and laser sources. 
     Referring to  FIG. 2 , in an embodiment, the optical coupler  14  comprises an optical medium  20  having a first surface  21  above which the sample is located. The use of the term “above” is for ease of description and is intended to differentiate between the side of the first surface  21  in which the light from the light sources  16  is directed (i.e. “below” the first surface  21 ) and the opposite side of the first surface  21  (i.e. “above” the first surface  21 ). It should be noted that only two light inputs  15  are shown for ease of illustration, however, generally the optical coupler  14  is configured to direct light received by the one or more light inputs  15  into the optical medium  20  in a manner suitable for creating an evanescent field above the first surface  21 . In an embodiment, the optical coupler  14  is configured to receive light from a plurality of light inputs  15  and to direct at least a portion of the received light towards the first surface  21  of the optical medium  20 . The evanescent field(s) located above the first surface  21  decay(s) exponentially with distance from the first surface  21 . Reference herein to evanescent field “penetration” refers to the distance from the first surface  21  for which the evanescent field is capable of interacting sufficiently with the sample  50  to produce an imageable or observable effect. Thus, where reference is made to increasing the penetration depth of the evanescent field, the reference means increasing the magnitude of the evanescent field at a particular (usually arbitrary) distance from the first surface  21 . 
       FIG. 2  also includes a representation of total internal reflection of light emitted towards the surface  21 . The figure illustrates two relevant angles: θ i  being the angle of incidence, and θ r  being the transmitted angle. The angles θ i  and θ r  are with respect to the is normal to surface  21 . The optical medium has a refractive index ni and the sample region has a refractive index n 2 . For angles of incidence θ i  larger than a critical angle θ c , total internal reflection will occur. The critical angle can be calculated according to: 
     
       
         
           
             
               
                 
                   
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     Each of the angles θ i , θ r , and θ c  are defined as the angle between the relevant ray and the normal to the surface  21  (as shown). For angles of incidence larger than the critical angle, θ i =θ r . 
     The sample  50  in a broad sense corresponds to features to be imaged by the imaging apparatus  10 . For example, the sample  50  can correspond to a single particle. In another example, the sample  50  comprises a plurality of individual particles. In yet another example, the sample  50  comprises surface features of a substrate—this can include surface features such as indents and protrusions. In a particular example, surface features of the first surface  21  of the optical medium  20  may be imaged (in this case, the optical medium  20  can also be considered the sample  50 ). The sample  50  is located within a sample medium, which, depending on the experimental arrangement, can be a vacuum (i.e. for the purposes of this disclosure, a vacuum is considered a type of sample medium) or air (i.e. with a refractive index n 2 ≈1), or can be any fluid or solid medium (as shown in  FIGS. 1 and 2 ) having a refractive index n 2 &gt;1. It is preferred that the sample medium has a refractive index lower than that of the optical medium  20  (i.e. n 2 &gt;n 2 ) to enable total internal reflection when light is incident on the surface  21  at angles of incidence exceeding the critical angle. Also, for the purposes of this disclosure and simplicity of language and unless otherwise noted, it is assumed that the sample  50  comprises a single particle. 
     It should be generally understood that the imaging sensor  13  can be configured to obtain a single image comprising all imaged features or a series of composite images which are subsequently combined to produce an image of the imaging region  11 . For example, in the latter case, individual light sources  16  are associated with unique wavelength spectra, and the imaging sensor  13  includes a plurality of sensors each uniquely associated with a light source  16  and configured to receive light associated with the light source  16 . In another example, a time series of composite images is combined to form the image. It is also understood that the image may be suitable for later decomposition into a plurality of composite images. 
     In an embodiment, the optical coupler  14  provides fixed angle coupling. That is, the coupling angle is fixed and selected such as to provide for total internal reflection for expected combinations of sample medium refractive index and optical medium  21  refractive index. In another embodiment, the optical coupler  14  provides modifiable angle coupling for each light input  15 . For example, mechanically or electrically actuated means may be provided for changing the coupling angle(s) for each light input  15  such as to enable variation in the angle(s) of incidence. In another example, moveable prisms may be utilised to adjust the direction of direction of the light into the optical medium  20 . This may be beneficial, for example, as it may enable adjustment of the angle of incidence to adjust the penetration depth and/or position of the evanescent field. 
       FIGS. 3A and 3B  show top-down views of the optical coupler  14  according to two embodiments. The optical coupler  14  includes a plurality of coupling points  23  for coupling to the light inputs  15 . In one embodiment, there are three coupling points  23   a - 23   c  as shown in  FIG. 3A . In another embodiment, there are more than three coupling points  23 , such as the eight coupling points  23   a - 23   h  shown in  FIG. 3B . Generally, it may be preferred that the coupling points  23  be located at equal spacings around the optical medium  20 . 
     Generally, each coupling point  23  is associated with a unique light input  15 . For example, in  FIG. 3A  there are light inputs  15 A- 15 C respectively associated with coupling points  23   a - 23   c  and in  FIG. 3B  there are light inputs  15 A- 15 H respectively associated with coupling points  23   a - 23   h.  In these embodiments, a light absorbing baffle region  24  is provided on the first surface  21  of the optical medium  20 . The baffle region  24  is configured to block light emitted from the light inputs  15 , which is incident upon the first surface  21  at an angle below the critical angle (θ c ), from entering the imaging region  11 . In one implementation, the baffling region  24  material is coloured black (or has a black appearance) as it is configured to absorb a broad spectrum of radiation. Typically, the baffle region  24  is utilised when the light emitted by the light inputs  15  is not collimated in order to reduce the amount of light incident at an angle below the critical angle (θ c ) that may be present in the imaging region. 
     For the purposes of this disclosure, a general feature shown in the figures is represented by a numerical reference—for example, the coupling points  23  of  FIGS. 3A and 3B . Such references, where appropriate, are common amongst the figures. Where reference to specific instances of a feature is desirable, a lowercase letter suffix is appended to the numerical reference—for example,  FIG. 3A  shows coupling points  23   a,    23   b,  and  23   c.    
       FIG. 3C  shows an embodiment wherein the light sources  16  themselves are components of the optical coupler  14 —typically, each light source  16  is secured within a body of the optical coupler  14 . The light sources  16  may be fixed within the optical coupler  14  or may be removable (such as to make the light sources  16  replaceable). In the implementation shown, the light sources  16  are LEDs, and there is a separate LED light source  16  for each light input  15 . Depending on the embodiment, the LED light sources  16  may emit different spectra or may emit the same spectra. An LED light source  16  can be configured to emit a single spectrum (i.e. a single “colour”) or a plurality of spectra (i.e. two or more “colours” as in the case of a multi-die LED). The LED light sources  16  may emit non-collimated light, and therefore, the optical coupler  14  according to the present embodiment will typically incorporate a baffle region  24 . The optical coupler  14  may include an electrical interface  25  enabling a power supply to be connected to each light source  16 . 
     The electrical interface  25  can be implemented as a single point of connection as shown or as multiple points (e.g. a connection point for each light source  16 ). 
     In an implementation of  FIG. 3C , each light source  16  produces substantially the same light spectrum. According to this implementation, each light source  16  is independently controllable such that each light source  16  can be in an emitting or non-emitting state independently of the other light sources  16 . For example, the electrical interface  25  can include a control line for each light source  16  as well as a common ground line (for example). In another implementation of  FIG. 3C , each light source  16  produces a unique light spectrum, which may be either continuous or discrete. According to this implementation, each light source  16  may be independently controllable such that each light source  16  can be in an emitting or non-emitting state independent of the other light sources  16 . However, in another option, each light source  16  is commonly controlled (i.e. each light source  16  is either emitting or non-emitting at the same time). For example, in this latter case, the electrical interface  25  can comprise a common control line as well as a common ground line (for example). The control lines may be connected to a controller which may be, for example, a computer  17  as described below. Alternatively, the controller may be a specifically programmed microcontroller, such as an ATMega 2560  microcontroller. 
     In an embodiment, the optical medium  20  of the optical coupler  14  includes a plurality of facets defining the sides of the optical coupler  14 , as shown in  FIG. 3D . In the example shown, there are six facets  54   a - 54   c  (three are obscured and not labelled in the figure), however, generally, any useful number of facets may be utilised. Light from light sources  16  (which may preferably be collimated) may be directed at the facets  54   a - 54   c . The optical coupler  14  of this embodiment defines a prismatic structure. The facets  54  can therefore be considered to be the light inputs  15 , with the angles of the facets  54   a - 54   c  and/or angle at which light is directed towards the facets  54   a - 54   c  selected such as to cause total internal reflection at the first surface  21 . The optical coupler  14  can include opaque regions as required (e.g. covering the first surface  21  except in a sample  50  location area of the first surface  21 ). 
       FIGS. 4A and 4B  depict the operation of an imaging apparatus  10  according to two embodiments, in each case without a sample being present within the imaging region  11 , with respect to the three-coupling points  23   a - 23   c  implementation of  FIG. 3A . In  FIG. 4A , each light source  16  (not shown in the figure) is associated with a unique spectrum—it may be preferred that the spectra are substantially non-overlapping—and therefore each light source  16  is activated simultaneously (indicated by the arrows). In  FIG. 4B , each light source  16  (not shown in the figure) produces the same (or similar) spectra, however, the light sources  16  are switched such that only one light source  16  is illuminated at a time.  FIG. 4B  shows illumination within the imaging region  11  at three separate time intervals (as indicated by the arrows). In an embodiment (not shown), each light source  16  is associated with both a unique spectrum and a unique point in time. In both  FIGS. 4A and 4B , each coupling point  23  is associated with a channel—in  FIG. 4A  channels are primarily characterised by the spectra of the light source  16  and in  FIG. 4B  channels are primarily characterised by the time period in which the corresponding light input  15  is activated. 
     As described with reference to  FIG. 1 , the imaging region  11  is imaged by the imaging sensor  13 , optionally via magnifier  12 . With no sample  50  present, it is expected that little to no light emitted by the light sources  16  is imaged by the imaging sensor  13 , due to the total internal reflection (it is understood that some of the emitted light may be imaged due to, for example, reflections within the optical coupler  14  or light leakage). 
     Referring to  FIGS. 5A and 5B , a sample  50  is shown present within the imaging region  11  of the optical coupler  14 . For the purposes of illustration, the sample  50  is shown as a simple sphere (i.e. having a circular cross-section as shown).  FIG. 5A  shows a collimated light rays  26   a,    26   b,    26   c  from one light source  16  being emitted into the optical medium  20  via light input  15 , and the light rays  26   a - 26   c  being reflected at the first surface  21 .  FIG. 5B  shows a non-collimated light rays  26   d,    26   e,    26   f,    26   g  from one light source  16  being emitted into the optical medium  20  via light input  15 , and light being reflected at the first surface  21 —as can be seen, each illustrated light ray  26   d - 26   g  follows a different path. Light ray  26   d  is following a path that would not result in total internal reflection at the first surface  21  but is absorbed (or at least substantially absorbed) by baffle region  24 . Light rays  26   e - 26   g,  however, do undergo total internal reflection at the first surface  21 . Of course, the light rays  26   a - 26   g  are merely illustrative. The baffle region  24  may be annular or may define a border mirroring the shape of the imaging region  11 . 
     A portion of the light incident onto the first surface  21  which would normally have undergone total internal reflection (e.g. in the case shown in  FIGS. 4A and 4B ) is in fact scattered by the sample  50 —the effect is as if the incident light is transferred via the evanescent wave to the sample  50 . Typically, the scattering is brightest (or strongest) at the surface boundary of the sample  50 . Thus, the total internal reflection is now frustrated due to the presence of the sample  50 . The resulting image shows a variation of detected intensity accord the imaging region  11 —this is referred to herein as an “intensity pattern”—which can be interpreted to determine one or more sample characteristics of the sample. 
     Without being bound to any particular theory, it is believed that an evanescent field generated in the region of the sample  50  interacts with the sample  50 , resulting in light emanating from the sample  50  that is correlated with the location of the surface(s) of the sample  50 . 
       FIG. 6A  shows an image  55   a  of a substantially spherical sample  50  with a diameter of approximately 5 microns. The sample  50  is illuminated by three light sources  16   a - 16   c  arranged with approximately equal angular spacing around the sample  50   a,  each light source  16   a - 16   c  associated with a different characterising spectrum.  FIG. 6C  shows an image formed by the imaging apparatus  10  with three light sources  16   a - 16   c  interacting with the sample  50   a  from three different angular directions. The resulting intensity pattern comprises diffraction patterns associated with each illumination direction, corresponding to approximately concentric rings surrounding a bright centre. In this case, sample characteristics such as the size and/or shape of the sample  50   a  can be estimated by determining and interpreting the relative location of the central “bright spot” for each of the three diffraction patterns. Thus, sample characteristics such as size parameters and/or shape parameters may be estimated despite diffraction limit considerations that may be applicable to directly imaging the sample  50   a.  It should be noted that the presence and extent of diffractive effects may be dependent on the spectrum of the light source(s)  16 , and the properties of the lenses of any magnifier  12  (for example, the numerical aperture). Under certain conditions for some samples  50 , local intensity variations resulting from a sample interacting with a single light source may be used to locate a plurality of surface features on a sample (for example local maxima or “bright spots” associated with at least two points on the surface of a sphere that may be used to determine radius). Inserts  1 - 3  show the individual bright portions of the intensity pattern of image  55   a.    
       FIG. 6B  shows an image  55   b  of another sample  50  having an irregular shape. However, the same principle applies as for the spherical particle  50  in  FIG. 6A —the intensity pattern can be directly or indirectly determined as corresponding to the surface portion at that location on the sample  50   b.    
     An intensity pattern can be characterised by an intensity pattern comprising local intensity maxima—i.e. the maximum intensity with a localised region. Such local intensity maxima can represent, for example in some cases, the actual location of a surface of the sample  50 . However, in other cases, the local intensity maxima are correlated with the location of the surface of the sample  50 , and a known adjustment can be applied to identify the surface location. It should be understood that the intensity pattern is a spatial pattern—it varies over the imaging region  11 . 
     Under certain conditions, sample characterises such as size parameters and/or shape parameters of a sample  50  may be determined by identifying and interpreting local intensity maxima—for example, the centre of each (or at least one or more, usually a plurality) bright region within the image  55  or the centre of each (or at least one or more, usually a plurality) bright region within a series of images of sample  50  (subject to the same or different evanescent field(s)) combined to form image  55 . 
     In  FIG. 6A , this is relatively trivial due to the nominally spherical shape of the sample  50 . The size of the sample  50  may be such that under certain conditions bright regions associated with a plurality of light sources  16  are relatively simple to isolate even when multiple light sources are simultaneously interacting with the sample  50  to produce an imageable effect, and no chromatic or temporal mechanism is in place to separate light emanating from the sample  50  according to the light source  16  associated with it. 
     In  FIG. 6A , this is relatively simple to account for as each bright region is substantively far enough away from each other bright region such that the approximate centre of each diffraction pattern can easily be identified. In  FIG. 6B , the bright regions can be interpreted with enough clarity to obtain an indication of shape parameters and size parameters of the sample  50  as diffractive effects are minimal. 
     Referring to  FIG. 6C , an image  55   c  is shown of a sample  50  with significant overlap in the diffraction patterns associated with three light sources  16 . A filtering procedure may be applied in order to isolate and identify each individual diffraction pattern and therefore the centre of each pattern. This filtering procedure may include physical techniques for separating light associated with each light source (for example via temporal sequencing or chromatic filtering) and may also include interpretation techniques applied during the analysis of resulting images. More generally, a filter may be utilised to assist in identifying is local intensity variations associated with edges/surfaces of the sample  50 . Alternatively, a user may utilise their judgement to interpret resulting images (for example when performing qualitative imaging, or when attempting to obtain an approximate estimate of a size parameter, or when attempting to determine the approximate shape of the sample  50 ). An advantage of an embodiment of the present description may be that it provides means to image or characterise small samples  50  which would otherwise be overly blurred due to diffractive effects. Inserts  1 - 3  show certain portions of the spatial intensity pattern of the image  55   c.    
     In an embodiment, the imaging sensor  13  is configured to continually (e.g. periodically) capture images of the imaging region  11  and to transmit the captured images to the computer  17 . The computer  17  includes a display  30  for displaying a graphical user interface (GUI). The GUI may be configurable to update on reception of a new image captured by the imaging sensor  13  to display the new image (typically the new image may replace a previously displayed image, or may be displayed alongside previously displayed images, or may be used to form a composite image based on a plurality of images of the imaging region  11 ). Therefore, the computer  17  can be effectively configured to display an up-to-date representation of the illuminated sample  50  and may also provide tools to analyse images of the imaging region  11 . 
     In an embodiment, the computer  17  or other control system is interfaced with the light input(s)  15  such that one or more output parameters of the light input(s)  15  can be adjusted through commands communicated from the computer  17  or other control system to the light input(s)  15 . For example, the intensity of the light inputs  15  may be adjustable in this manner. Furthermore, the on/off state of the, or each, light input  15  may be set by a command sent from the computer  17  or control system. 
     In another embodiment, the light input(s)  15  are interfaced with a controller  19  which is configured to control one or more parameters of the light input(s)  15 . The controller  19  itself may be interfaced with the computer  17  such that the computer  17  causes the controller  19  to operate in a particular manner. For example, the controller  19  may automatically control the light input(s)  15  in accordance with a mode selected from a plurality of modes by the computer  17 . 
     In a more general sense, in an embodiment, the computer  17  is configured to control the light input(s)  15  and/or the light sources  16  and/or the imaging sensor  13  to implement the processes described herein. The computer  17  can be a general-purpose computer interfaced with a controller  19  or can be directly interfaced with the relevant components of the system  10 . 
     In an embodiment, the imaging sensor  13  corresponds to an RBG sensor (e.g. the imaging sensor  13  is a component of a digital camera that may be mounted to a magnifier  12 ). The RGB sensor may be particularly useful for imaging a sample  50  where the light sources  16  comprise a plurality of substantially red, blue, or green wavelengths—for example, each light source  16  may have the substance of its spectrum selected to overlap preferentially with detection sensitivity of one or more particular elements of the colour filter array of the RGB sensor. The use of an RGB sensor and corresponding light sources  16  may then provide an advantage in that the image(s) produced by the imaging sensor  13  may in some cases be more easily decomposed to associate bright regions of distinct substantive wavelengths with their respective light sources  16 . 
     In some cases, cross-talk between adjacent sub-pixels of the RGB sensor (for example, an RGB sensor with Bayer configuration) may introduce error when determining sample characteristics such as size parameters and/or shape parameters of a sample  50 . According to an embodiment, a deconvolution filter may be applied to the signal generated by the RGB imaging sensor  13  (e.g. the image data obtained corresponding to the image  55 ). The deconvolution filter may be configured based on known cross-talk properties of the RGB sensor (e.g. the sensitivity of a red sub-pixel to the relevant spectra of red, green and blue incident light) to calculate (or estimate) the light associated with each of the relevant light sources  16  incident on at least one physical pixel of the imaging sensor  13 , approximately independent of other spectra also incident on the same physical pixel(s) of the imaging sensor  13 . 
       FIG. 7  shows a method for operating the imaging apparatus  10  to take measurements of a sample  50  positioned on a sample substrate  51  and within a sample medium  52  (which may be vacuum, air, or some other medium). The sample  50  is located within the imaging region  11 . An initial calibration step  100  may be required, as shown in  FIG. 7 . The calibration step may include, for example, making necessary focusing adjustments to any magnifier  12  that may be used. According to the present embodiment, each light source  16  may be associated with a unique characteristic spectrum (e.g. refer to  FIG. 4A ). At illumination step  101 , the optical medium  20  is illuminated with at least two of the light sources  16  simultaneously. As previously described, it is expected that most of the incident light will undergo total internal reflection at the surface  21 . In an embodiment, ideally only totally internally reflected light rays would be propagated through the optical medium. In proximity to the sample  50 , light incident on surface  21  at an angle matching or exceeding the critical angle for total internal reflection at this surface may undergo frustrated total internal reflection, and a portion of the incident light may be transmitted to surfaces of the sample  50  producing scattering from the sample  50 . 
     At imaging step  102 , an image of the imaging region  11  is captured using the imaging sensor  13 . The imaging sensor  13  may be coupled to a computer  17  (or other processing device) and/or directly to a storage medium such as a non-volatile memory  18  (e.g. a FLASH memory). The imaging sensor  13  is configured such as to allow for differentiation in the captured image between each of the characterising spectra—for example, the imaging sensor  13  may comprise an RGB sensor. The captured image therefore includes information indicating light intensity and light wavelength. The image  55  is then analysed at analysis step  200  (described in more detail below). 
       FIG. 8  shows another method for operating the imaging apparatus  10  to take measurements of a sample  50  positioned on a sample substrate  51  and within a sample medium  52  (which may be a vacuum, air, or some other medium). The sample  50  is located within the imaging region  11 . An initial calibration step  110  may be required, as shown in  FIG. 7 . The calibration step may include, for example, making necessary focusing adjustments to any magnifier  12  that may be used. According to the present embodiment, each light source  16  is associated with a temporal position. At illumination step  111 , the optical medium  20  may be illuminated with at least one of the light sources  16 . As previously described, it is expected that substantially all of the incident light will undergo total internal reflection at the first surface  21 . In proximity to the sample  50 , light incident on surface  21  at an angle matching or exceeding the critical angle for total internal reflection at this surface may undergo frustrated total internal reflection, and a portion of the incident light may be transmitted to surfaces of the sample  50  producing scattering from the sample  50 . 
     The methods of  FIG. 7  and  FIG. 8  may effectively be combined, such that a sequence of illuminations occurs wherein at least one step in the sequence comprises at least two light sources illuminating the optical medium  20  simultaneously with different unique characterising spectra. For example, a sequence of illuminations may occur where, in each step in the sequence, the optical medium  20  is illuminated with three light sources  16  corresponding to red, green, and blue. Preferably, each illumination is from a unique angle. For example, if each sequence step illuminates with three light sources  16 , and there are four steps in the sequence, the optical medium  20  is illuminated from twelve different directions (viz. four sequence steps multiplied by three angles of illumination per step). 
     At imaging step  112 , an image of the imaging region  11  is captured using the imaging sensor  13 . The imaging sensor  13  may be coupled to a computer  17  (or other processing is device) and/or directly to a storage medium such as a non-volatile memory  18  (e.g. a FLASH memory). The captured image is associated with the temporal position. 
     At step  113 , the method checks whether all of the light sources  16  have been imaged. If not, the method moves to step  114  wherein a new light source  16  is selected and then back to step  111  where the imaging region  11  is illuminated with the newly selected light source  16 . Step  112  is repeated with the captured image being associated with the new temporal position associated with the newly selected light source  16 . If yes, then the images  55  are then analysed at analysis step  200  (described in more detail below). 
     Step  200  corresponds to analysis of the image(s)  55  obtained according to  FIG. 7 or 8 . Generally, whether one image  55  or multiple are captured, the analysis may proceed on the basis that each light source  16  may illuminate the sample  50  from a different direction. Thus, each of the scattering maxima (or other distinguishing intensity variations) associated with the sample  50  is associated with a particular light source  16  and direction. There may be overlap in scattering patterns associated with different light sources  16 . Multiple images  55  may be combined into a single image  55  and with artificial colouring (for example) may be assigned to the contribution of each image. 
     According to an embodiment, under certain conditions sample characteristics such as size parameters and/or shape parameters of a sample  50  may be determined by identifying and locating imaged maxima—this can be performed for example by a user viewing the image  55  or using software configured to identify such maxima. 
     According to an embodiment, where diffractive or light-scattering effects must be accounted for, an analysis may proceed on the basis of local maxima of light intensity in an image  55 . A suitable filter or processing step may be required in order to accurately characterise different local maxima—for example, one or more parameters may be adjusted in order to determine the position of local maxima within the image(s)  55  relative to the size parameter(s) and/or shape parameter(s) of sample  50 . These local maxima correlated with the surface (or at least one or more regions of the surface) of the sample  50 . A user may be enabled to identify sample  55  sample characteristics such as size parameters and/or shape parameters, or software may be utilised for this purpose. 
     In a more general sense, the obtained image data may be put through an equipment processing filter step. The purpose of this step is to modify the data based on known properties of the imaging apparatus  10 . For example, diffractive effects may be accounted for based on known properties of the detector optics (e.g. the numerical aperture of an objective). Also as previously discussed, account may be taken of any cross-talk between colour sub-pixels of the colour filter array in an RGB imaging sensor  13 . 
     Furthermore, a sample processing filter step may be utilised. The purpose of this is to account for known properties of the sample  50 . For example, it may be known that for a particular sample  50 , the local maxima are displaced with respect to a sample surface. The step may therefore account for this by effectively “moving” the determined sample surface, thereby affecting a subsequent interpretation and characterisation of the sample  50 . 
     Sample characteristics such as shape parameter(s) and/or size parameter(s) of the sample  50  may be determined based on the local maxima as shown in the image when accounting for the magnification of any magnifier  12  that may be used, and the resolution of the imaging sensor  13 . From these details, distances may be determined within the image between local maxima. 
     In an embodiment, as shown in  FIG. 9 , the sample  50  comprises particles contained within a fluid  53  (e.g. a gas such as air or a liquid such as water), where the fluid  53  is moving with respect to the imaging region  11 —therefore, the particles are also moving with respect to the imaging region  11 . The fluid  53  may be contained within guide means (e.g. a pipe or fluid cell)—this is not shown. The imaging apparatus  10  may be utilised to obtain particle characteristic measurements of the particles. The light inputs  15  may be strobed—that is, the illumination time of each light input  15  is relatively short such that the average movement of the particles may small enough to enable accurate particle characterisation during illumination. In embodiments such as that shown in  FIG. 4B , the sequence of light input  15  illumination may also be relatively short, such that during the entire sequence the movement of the average particle movement is short. Preferably, each image captured by the imaging sensor  13  corresponds to a single illumination or sequence of illuminations. In embodiments such at that shown in  FIG. 4B , light input(s)  15  may comprise a single spectrum or a plurality of spectra, and may operate for example in either of the modes depicted in  FIG. 7  or  FIG. 8 , or some combination of these or other modes of operation. 
     Similarly, in an embodiment, the sample  50  is suspended in a liquid such that the sample  50  may move (e.g. due to Brownian motion) while contained above the first surface  21 . Again, a strobing approach may be utilised such that the illumination time of the sample  50  is short to ensure minimal displacement of the sample  50  during image acquisition. 
       FIG. 10  shows an embodiment having a single light source  16  coupled to a rotating light input  15  of a rotating ring  28 . The ring  28  is located between a prism  29  and the light source  15 . The single light source  16  is directed through a lens and/or mirror arrangement  27  which is configured to produce an annular light pattern  30  (as shown), which has a diameter corresponding to the diameter of the ring  28 . The light input  15  corresponds to an aperture  31  in the ring  28 . Light exiting the light input  15  is refracted by a prism  29  such that it is directed, at an angle suitable for total internal reflection, into the optical medium  20 . Therefore, the angle from which the sample  50  is illuminated may be effectively continuously changes in accordance with the rate of rotation of ring  28 . 
     Embodiments described herein may provide for measurement of the sample characteristics such as size and shape of individual particles of a sample  50 , in air or liquid. The embodiments may advantageously be useful for feature sizes (such as particles) having a size in the range of 1 millimetre (or maybe larger) down to  10   s  of nanometres (or maybe smaller). The techniques described may be suitable for building particle size parameter and/or shape parameter distributions from a large number of single-particle measurements. 
       FIG. 11  shows an illustration of a composite image  61  and the four channel images  60   a - 60   d  that make up the composite image  61 . That is, channel images  60   a - 60   d  each correspond to a direction of illumination and, in this case, a particular unique spectrum. The different channel images  60   a - 60   d  show the effect of illuminating the sample from different directions with evanescent fields, and the composite image  61  shows how the sample  50  may be characterised e.g. in determining shape parameters and/or size parameters. The optical image  62  shows that the correspondence between the composite image  61  and actual particle shape is consistent. 
       FIG. 12  shows an example image  55   d  of a plurality of particles making up the sample  50 . It may be possible to characterise each of these particles. It may also be possible to determine statistical features such as average shape parameter(s) and/or size parameter(s). 
       FIG. 13  shows an example image  55   e  of a plurality of particles making up the sample  50 . In this case, there is included a larger particle (see broken line square box) and several smaller particles—the insert shows a magnified image of the smaller particles. Advantageously, the embodiments herein described may be suitable for identifying characteristics of particulars with large differences in size (e.g., in this case, roughly an order of magnitude) in a single imaging procedure. 
     Further modifications can be made without departing from the spirit and scope of the specification. For example, as mentioned herein, in some cases there may be a visible scattering bright region opposite a light input  15  in addition to a scattering bright region adjacent the light input  15 . Where a geometry of a sample  50  is known to be relatively or substantially symmetrical (e.g. a sphere), a single illumination direction (i.e. one light input  15 ) may be sufficient to characterise the sample  50 . In another example, where the pattern of scattered light resulting from at least one illumination direction and spectrum is well known for the type of sample  50  under observation, a single illumination direction (i.e. one light input  15 ) may be sufficient to characterise the sample  50  by reference to an existing database or model. Although size and shape parameters have been described herein for the purposes of exemplification, it is also anticipated that the resulting image when imaging a sample  50  may be interpreted to identify other sample characteristics—this may depend, for example, upon known properties of the sample  50  (e.g. a model developed through previous experimentation on the sample  50 ). Such a variation may be particularly useful for identifying a type of material present in the sample  50 . In one example, a size or shape of a sample  50  may correlate with a particular material expected to be present within the sample  50 . 
     Reference herein to background art is not an admission that the art forms a part of the common general knowledge in the art, in Australia or any other country.