Apparatuses for analyzing the optical properties of a sample

Abstract

A method of analysing a sample in the form of a droplet provided on a sample-receiving surface includes providing a light source and a detector in a housing, positioning said sample-receiving surface in or on the housing, and focussing an incident beam of light to a focal point in the vicinity of the sample. Light is detected from the sample resulting from an interaction with the sample, the sample-receiving surface, or the atmosphere surrounding the sample. At least one parameter of the detected light is measured, and the sample-receiving surface is translated relative to the housing such that the focal point is at a different region of the sample, the sample-receiving surface, or the atmosphere surrounding the sample. The step of measuring one or more parameters of the detected light is repeated following the translating step.

Claims

1. A method of analyzing a sample in the form of a droplet provided on a sample-receiving surface, the method comprising: providing a light source and a detector in a housing; positioning said sample-receiving surface in or on the housing; focussing an incident beam of light to a focal point in the vicinity of the sample; detecting light from the sample resulting from an interaction with the sample, the sample-receiving surface, or the atmosphere surrounding the sample; measuring one or more parameters of the detected light; translating the sample-receiving surface relative to the housing such that the focal point is at a different region of the sample, the sample-receiving surface, or the atmosphere surrounding the sample; repeating the step of measuring one or more parameters of the detected light following said translating step; analyzing a plurality of measurements of the one or more parameters of the detected light to determine an optimal measurement position; and performing a fine scan by repeating the translation of the sample-receiving surface in the vicinity of said optimal measurement position to obtain an improved measurement position.

2. The method of claim 1, wherein the steps of translating the sample-receiving surface and repeating the step of measuring the one or more parameters of the detected light are performed multiple times, to provide a plurality of measurements taken from different positions relative to the sample.

3. The method of claim 2, wherein said plurality of measurements are taken throughout the sample volume.

4. The method of claim 2, wherein said plurality of measurements comprise a series of measurements taken in a plane parallel to the sample-receiving surface, repeated at additional parallel planes in a plurality of slices through the sample volume.

5. The method of claim 1, wherein the step of translating the sample comprises translating the sample in a plane parallel to the sample-receiving surface.

6. The method of claim 5, wherein the step of translating the sample comprises translating the sample in two dimensions such that a plurality of measurements are taken across said plane.

7. The method of claim 1, further comprising: translating the sample-receiving surface in a direction normal to the sample-receiving surface and repeating a plurality of measurements in another plane located at a different distance from the sample-receiving surface.

8. The method of claim 1, further comprising: translating the focal point in a direction normal to the sample-receiving surface and repeating a plurality of measurements in another plane located at a different distance from the sample-receiving surface without translating the sample-receiving surface in a direction normal thereto.

9. The method of claim 1, wherein the sample-receiving surface is provided with nanostructures enabling a surface enhanced Raman spectroscopy (SERS) response, and the optimal measurement position provides a maximal SERS signal.

10. The method of claim 1, wherein analyzing the plurality of measurements comprises aggregating or integrating said plurality of measurements.

11. The method of claim 1, further comprising the steps of: obtaining an image of the sample using an imaging device; determining one or more sample boundaries from said image; and determining one or more translation limits within which the sample-receiving surface is to be translated to thereby enable a plurality of measurements to be taken from desired portions of the sample, the sample-receiving surface or the atmosphere surrounding the sample.

12. A method of analyzing a sample in the form of a droplet provided on a sample-receiving surface, the method comprising: providing a light source and a detector in a housing; positioning said sample-receiving surface in or on the housing; focussing an incident beam of light to a focal point in the vicinity of the sample; detecting light from the sample resulting from an interaction with the sample, the sample-receiving surface, or the atmosphere surrounding the sample; measuring one or more parameters of the detected light; translating the sample-receiving surface relative to the housing such that the focal point is at a different region of the sample, the sample-receiving surface, or the atmosphere surrounding the sample; repeating the step of measuring one or more parameters of the detected light following said translating step; obtaining an image of the sample using an imaging device; determining one or more sample boundaries from said image; and determining one or more translation limits within which the sample-receiving surface is to be translated to thereby enable a plurality of measurements to be taken from desired portions of the sample, the sample-receiving surface or the atmosphere surrounding the sample, wherein the step of determining one or more translation limits further comprises determining limits within which the focal point is to be translated in a plane normal to the sample-receiving surface.

13. A method of analyzing a sample in the form of a droplet provided on a sample-receiving surface, the method comprising: providing a light source and a detector in a housing; positioning said sample-receiving surface in or on the housing; focussing an incident beam of light to a focal point in the vicinity of the sample; detecting light from the sample resulting from an interaction with the sample, the sample-receiving surface, or the atmosphere surrounding the sample; measuring one or more parameters of the detected light; translating the sample-receiving surface relative to the housing such that the focal point is at a different region of the sample, the sample-receiving surface, or the atmosphere surrounding the sample; repeating the step of measuring one or more parameters of the detected light following said translating step; obtaining an image of the sample using an imaging device; determining one or more sample boundaries from said image; and determining one or more translation limits within which the sample-receiving surface is to be translated to thereby enable a plurality of measurements to be taken from desired portions of the sample, the sample-receiving surface or the atmosphere surrounding the sample, wherein the sample boundaries define a three-dimensional volume.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will now be further illustrated by the following description of embodiments thereof, given by way of example only with reference to the accompanying drawings, in which:

(2) FIG. 1 is a perspective view from a first angle of an apparatus according to the invention;

(3) FIG. 2 is a perspective view from a reverse angle of the apparatus of FIG. 1;

(4) FIG. 3 is a perspective view of a sample holder assembly of an apparatus of the invention;

(5) FIG. 4 is a perspective view of the assembly of FIG. 3 with the cover open;

(6) FIG. 5 is an exploded view of the FIG. 3 assembly;

(7) FIG. 6 is a top view of the FIG. 3 assembly;

(8) FIG. 7 is a sectional elevation of the FIG. 3 assembly taken along the line B-B in FIG. 6;

(9) FIG. 8 is an enlarged detail of the sample holder with the cover of the FIG. 3 assembly closed;

(10) FIG. 9 is a detail of the assembly of FIG. 3 from which FIG. 8 is taken;

(11) FIGS. 10A-10C are respectively a side elevation, a perspective view from above, and a top plan view of a sample holder;

(12) FIG. 11 is a perspective view of the sample holder assembly of FIG. 3 with a SERS plate in place of the sample holder;

(13) FIG. 12 is a sectional elevation through the assembly of FIG. 11;

(14) FIG. 13 is an exploded view of the assembly of FIG. 11;

(15) FIG. 14 is an exploded view of an alternative configuration of sample holder assembly with a vial holder in place of the sample holder;

(16) FIG. 15 is a perspective view of an alternative apparatus prior to mounting the sample holder assembly and the spectroscopic assembly;

(17) FIG. 16 is an exploded view of a first spectroscopic assembly;

(18) FIG. 17 is an exploded view of a second spectroscopic assembly;

(19) FIG. 18 is a detail of a further embodiment;

(20) FIG. 19 is a schematic illustration of a scanning technique for obtaining multiple measurements from a sample;

(21) FIG. 20 is a schematic illustration of a further scanning technique for obtaining multiple measurements from a sample;

(22) FIG. 21 is an embodiment adapted to perform scanning techniques such as are shown in FIGS. 19 and 20;

(23) FIG. 22 is a view from above of a mechanism for translating a cover assembly;

(24) FIG. 23 is a view from below of the FIG. 22 mechanism;

(25) FIG. 24 is a sectional schematic view of a further apparatus with a different mechanism for bringing a sample into position for measurement;

(26) FIG. 25 is a perspective view of a sample holder (plinth).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(27) In FIGS. 1 and 2, there is indicated, generally at 10, an apparatus for analysing the properties of a liquid sample. The apparatus 10 comprises an enclosure 12 defining an internal space 14 which receives a spectroscopic assembly 16 having a source and a detector behind an optical port 18.

(28) The spectroscopic assembly 16 is mounted on a cover plate 20 having securing fasteners 22 which secure the cover plate to the housing. The vertical (or Z) position of the cover plate and hence the spectroscopic assembly can be adjusted by a micrometer adjustment mechanism acting against a stop 26 on the housing, with slots 28 in the cover plate accommodating a range of vertical positions.

(29) An evaporation protection tray 30 can be mounted in the housing. When compartments in this tray are filled with a solvent (such as alcohol or water) this acts as a reservoir to ensure that the interior of the housing becomes saturated with vapour, which in turn inhibits evaporation of a small volume sample exposed to the interior of the housing. Adjustable feet 32 permit the apparatus to be accurately levelled.

(30) A sample holder assembly 34 is mounted on top of the housing and comprises a base plate 36 and a hinged cover 38, the base plate being mounted in a recess 40 on the top surface of the housing. The construction of various sample holder assemblies will be described in more detail below.

(31) FIG. 3 shows one embodiment of the sample holder assembly, which has the base plate 36 and cover 38, the cover being mounted on a hinge 42 which allows the cover to be lifted by a handle 44 and flipped backwards to reveal the underside or interior face of the cover and the area of the base plate concealed by the cover. A pair of securing screws 46 are shown in FIG. 3 for securing the base plate to the housing. Also shown are a first micrometer adjuster 48 and second micrometer adjuster 50 which permit fine adjustment of the cover position in X and Y directions relative to the base.

(32) FIG. 4 shows the sample holder assembly of FIG. 3 when the cover 38 has been flipped back through about 180 degrees about the hinge 42. Referring additionally to FIG. 5, this shows the same components, but in exploded view.

(33) It can be seen from FIGS. 4 and 5 that the base plate 36 accommodates a vapour tray 52. The vapour tray may render redundant a large vapour tray in the housing and it serves to provide a small enclosed volume within which a sample will sit when the cover is closed. The vapour tray will be described further below, but it suffices to note that it contains a glass window that exposes the sample to the optical components of the spectroscope.

(34) The sample itself is not shown but FIG. 4 shows the sample holder 54 in exploded view. The sample holder is in the form of a stainless steel body having a raised central disk, platform or plinth 56 which is sized to receive a small volume of sample in the form of a liquid or semi-solid (which term includes gels, pastes and slurries) volume that is sufficiently small to be retained on the plinth by surface tension or Laplace pressure when the plinth is inverted (as when the cover is closed).

(35) The stainless steel sample holder 54 is received in a plinth holder 58 that is mounted in the cover by a grub screw 60. A bore 62 (which is not fully visible in the view of FIG. 4 extends radially through the plinth holder 58 from the external annular surface to the internal annular surface.

(36) A retaining spring member 64 is located in the bore 60 prior to mounting the plinth holder in position in the cover and securing it with the grub screw. The retaining spring member has a cylindrical body with an internal spring-mounted cylinder whose tip 66 extends from the inner end thereof such that in use it protrudes into the central hole of the plinth holder. The inner cylinder can be forced back into the cylindrical body of the retaining spring member 64 against spring pressure, and in this way it serves to accommodate and secure the sample holder 54 in the plinth holder 58.

(37) This permits different sample holders such as sample holder 54 to be swapped into and out of the plinth holder 58 using a custom-designed tool 68 that is adapted to engage the rim of the sample holder without contacting the central plinth or a sample loaded thereon.

(38) It can be seen that the part of the hinge 42 which rotates with and is integral with the cover has a recessed groove or cam surface 70 which registers with a micro switch 72 when the cover is closed. Opening the cover is thereby detected by the micro switch and this is connected to the spectroscope to disable it. When the cover is closed the micro switch detects this and enables the operation of the spectroscope. This is an important safety feature when lasers are used in the spectroscope, and it can also protect sensitive detectors.

(39) Also shown in FIG. 5 are a back cover plate 74 for covering the rear of the plinth holder and a vial holder clamp 76. The back cover plate 74 may be removed, along with the plinth holder 58 and the sample holder 54, and an alternative form of sample holder, such as a vial, to be mounted to the vial holder clamp, in a modified form of operation.

(40) FIG. 6 shows a top plan view of the sample holder assembly of FIGS. 3-5 (with internal features shown in broken lines), and FIG. 7 shows the cross-section through FIG. 6 taken along the line B-B.

(41) It can be seen that with the cover 38 in its closed position the plinth holder 58 is brought face-down onto the vapour tray 52.

(42) FIGS. 8 and 9 show the detail of this arrangement in enlarged form, with FIG. 8 being a detail of FIG. 9. The vapour tray 52 has an internal annular moat 78 and a raised central cylindrical wall 80 that defines the internal wall of the moat 78. The moat can be filled with a solvent (such as the solvent used in a droplet of sample (not shown) positioned and retained on the central plinth 56), and this promotes a saturated atmosphere to inhibit evaporation of the droplet in the very small confined volume between the cover and the vapour tray when the cover is closed.

(43) The plinth is positioned centrally over the cylindrical wall 80, which in turn is aligned vertically with the optical port of the spectroscope, allowing light to illuminate the sample and for light from the sample (e.g. reflected, refracted or emitted radiation) to be collected. The underside of the vapour tray is separated from the rest of the interior of the housing by a transparent (e.g. quartz glass) disc 82 to maintain separation of the atmospheres while maintaining optical continuity. It will be appreciated that such a cover need only be transparent to wavelengths of interest, which may vary according to the type of analysis being conducted and the wavelengths used (e.g. infrared, visible or ultraviolet spectroscopy, Raman spectroscopy or fluoroscopic measurements). A sample located on the plinth, in this way, is brought to a predefined sample locus where measurements can be taken reliably and accurately using the source and detector which are also in predetermined positions.

(44) FIGS. 10A-10C show an alternative form of sample holder 54′. As with the holder shown in FIGS. 4-9, a central raised plinth 56 is provided to receive a sample thereon. Also as in the earlier embodiment, a rim 84 extends peripherally from the top surface 86 and this allows engagement of the sample holder by the tool 68 (FIG. 4). Unlike the sample holder seen in FIG. 4, however, the sample holder 54′ of FIGS. 10A-12 has a circumferential V-shaped recess 88 around its cylindrical surface. This recess can engage with the retaining spring member 64 of FIG. 4 regardless of the rotation of the sample holder when it is inserted into the plinth holder. In contrast the sample holder 54 of FIG. 4 can be seen to have a specific recess positioned on its cylindrical surface, requiring it to be inserted with the correct orientation to be properly retained.

(45) While the sample holders thus far described are machined from a single piece of stainless steel, alternative materials can be used. In particular, quartz and ceramic holders are very suitable. Such holders can be made entirely from an alternative material, or for example a quartz or ceramic rod having the diameter of the plinth only may be embedded centrally in a larger sample holder body made from any suitable material (such as metal, glass or plastic).

(46) The provision of the sample holder (whether of FIGS. 4-9 or of FIGS. 10A-10C or any alternative version) on the internal surface of the cover, and the flipping motion of the cover whereby it can be moved in a hinged manner between closed and opened positions, provides significant advantages. It enables small (microliter-sized) droplets and samples to be accurately and easily positioned on a very small target area with the target area exposed and fully accessible

(47) FIGS. 11-13 shows an embodiment similar to that of FIGS. 4-9 in the operation and movement of the cover, but in which the sample holder is a SERS plate 90 carried by the cover instead of the sample holder 54 or 54′. SERS (or surface enhanced Raman spectroscopy) is a technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces or customised nanostructures. The enhancement factor can be as much as 10.sup.10 to 10.sup.11, which opens up the possibility of single molecule detection.

(48) SERS arose out of the observation that a roughening of the surface the molecules are adsorbed on helps enhance the Raman process, which in fact has proven to be a result of enhancement of the local electromagnetic field close to the roughened surface due to the excitation of a localized surface plasmon, and chemical mechanisms giving rise to further enhancement for molecules adsorbed onto specific sites (hot-spots) where the resonant charge transfer occurs. The resulting enhancements occur most commonly when the excitation laser frequency coincides with a localized surface Plasmon resonance (LSPR) of a plasmonic-active material such as Au, Ag and Cu, over normal Raman scattering, making it a very powerful technique for sensing, as it also offers label-free detection of biomolecules in their natural environment. In the study of biological species, SERS is often coupled with resonance Raman by using a laser excitation wavelength in resonance with the molecular electronic levels (SERRS).

(49) The complexity of SERS, particularly in relation to sample preparation—that may also factor in the SERS background—is a current bottleneck to realisation of its full potential. SERS has been shown to be capable of single molecule detection with high specificity, but single-molecule sensitivity in diluted solution is a challenge which limits its application, particularly in forensics and healthcare diagnostics, where for example, early warning focuses on small concentrations of biomarkers in biofluids such as blood. SERS depends on the statistical binding of analytes to the SERS hot-spots where the electromagnetic field is particularly intense. The creation of such hot-spots is a highly-active area of research in the field, with various approaches to nanofabrication being taken, including top-down lithography-based fabrication, and bottom-up wet chemistry of structures on surfaces. In the liquid phase, molecules can be dispersed far away from the surface, making an encounter of the molecules with hot-spots statistically unlikely. Approaches to overcoming this problem include the introduction of plasmonic nanoparticles into the solution, functionalisation of nanoparticles with ‘grabby’ bioreceptors in the case of biomolecules, the use of microfluidic channels, nanoporous membranes, super-hydrophobic surfaces and external—both electrical and optical—stimuli to guide and concentrate the molecules towards the fabricated hot-spots. Some approaches use a combination of techniques to increase the possibility of an analyte interaction with a hot-spot.

(50) The plate 90 is prepared in advance with a suitable surface treatment, such as gold or silver nanoparticle colloidal preparations. The sample is positioned on the SERS surface, and measurements are taken of the surface enhanced Raman spectrum. In an alternative modification, the SERS plate can be mounted on the base plate when the cover is opened to reveal the base plate. A raster scanning mechanism can be provided to move the illumination spot from a laser in the spectroscope (whether by physically moving the laser, or the spectroscope assembly, or by optically scanning the position from a fixed laser in known manner). In this way the return signal can be detected to determine a maximum when the illumination spot is targeted to a point of maximum SERS activity on the SERS plate, and this can be used as a pre-calibration step.

(51) FIG. 14 shows a modification to the embodiment of FIGS. 3-9, in which the sample holder 54, plinth holder 58 and back cover 74 have been removed and in their place a vial holder 91 has been mounted, using the vial holder clamp 76, to the outside of the cover 38, with a sample-containing vial 92 exposed to the interior of the cover. A protective cap 94 is provided on the exterior of the vial holder 91. This modification adapts the instrument for measurement of samples in vials rather than in the form of droplets or small samples on a sample-receiving surface.

(52) FIG. 15 shows an alternative embodiment of apparatus from FIG. 1, with the sample holder assembly 34 of FIG. 14 ready to be mounted in place. It will be appreciated that this is the same overall sample holder assembly as in FIGS. 3-9 in terms of its construction, base plate and cover, and only the vial holder differentiates it from the FIGS. 3-9 embodiment. The sample holder assembly 34 is mounted in place onto a recess 96 on the top surface 98 of a housing 100. A removable access plate 102 enables a user to access the interior for removal and replacement of a spectroscopic assembly 104 (shown outside the housing). As in FIGS. 1 and 2, this assembly 104 is mounted on a mounting plate 106 which can be fixed into position within the housing with brackets 108 (FIG. 16) and thereby correctly position the assembly 104 relative to the sample locus. A spacer 110 adapts the position of a particular spectroscopic assembly to the mounting plate 106, and it will be appreciated that different instruments can thereby be substituted into the correct position.

(53) The spectroscopic assembly pictured is a CBEx handheld Raman spectrometer (produced by Snowy Range Instruments of Laramie, Wyo.) which provides a spectral range of 400 to 2300 cm-1 from a 70 mW laser through an optical port 112, with the ability to perform raster scanning. Any other suitable spectroscopic instrument can be used.

(54) FIG. 17 shows a fibre optic Raman probe 114 which is connected by fibre optic cable 116 to an external spectroscope (not shown), the optical signals being emitted and received by an optical port 118 which acts as source and detector. As with the embodiment of FIG. 16, the probe 114 is mounted on a plate 120 which is mountable within the housing 100 of the FIG. 15 apparatus. Both the FIG. 16 and FIG. 17 embodiments have a micrometer adjustment mechanism 122 for varying the vertical Z position of the spectroscopic assembly relative to the housing.

(55) The above embodiments describe a free-space optics implementation, where the source is remote from the sample, and light is transmitted to the sample from the source and from the sample to the detector within the apparatus.

(56) FIG. 18 shows a detail of an alternative embodiment, in which the source and detector (not shown) are each coupled to an optical fiber 130, which terminates at a terminal surface 132. The terminal surface 132 is positioned relative to the sample-receiving surface 134 (shown when the cover 138 is closed) such that the sample 136 makes contact with the terminal surface 132. The contact may be with the surface of the sample as shown or the terminal surface may be positioned such that it is immersed in the sample.

(57) The arrangement couples the light directly into and out of the sample. The terminal surface 132 may be treated with a surface treatment that provides an enhanced response, such as a nanostructured coating or treatment that provides a SERS response. The terminal surface or some other part of the optical fiber that is immersed in the sample may be coated with a reagent or active agent that causes a chemical reaction or a biological response, enabling the sample to be analysed by generating such a reaction or response and analysing a spectroscopic characteristic of the resultant species.

(58) FIG. 18 shows the terminal surface as integral with the optical fiber. The terminal surface may alternatively be provided on an end member or cap that is placed on the optical fiber and is transparent to a frequency of interest. This permits the cap to be easily swapped in and out, which may be advantageous to enable different responses and reactions to be studied, or to avoid cross-contamination between samples.

(59) FIG. 19 shows a schematic illustration of a scanning capability that may be incorporated in the apparatuses described herein. A camera 150 is positioned to image a sample 152 (in this case a droplet) positioned on the sample-receiving surface of a plinth 154. It will be appreciated that the sample is shown sitting on top of the surface but in the embodiments described above this arrangement is typically inverted so that the droplet hangs upside down from the sample-receiving surface when the cover is in the closed position. Light 158 from a source 156 is focussed (by the optical properties of the source and the refractive properties of the droplet) to a point 160 on the surface where the droplet sits.

(60) By translating the sample-receiving surface in two dimensions, e.g. using an x- and y-raster scanning mechanism, the detected signal may be integrated across the surface, or a search may be performed for an optimum signal position. In the case of SERS measurements, where the response may increase by several orders of magnitude at points where the excitation frequency coincides with a localized surface Plasmon resonance (LSPR) of a plasmonic-active material such as Au, Ag and Cu, there may be localized hot spots that provide greatly increased signal strength.

(61) Accordingly, the surface may be translated through a scanning pattern of any suitable design (for example an x-y raster pattern, an orbital pattern or any other suitable pattern), to identify areas of increased response, using feedback from the detector. This is preferably done as a coarse search, followed by a fine scan in one or more areas of particularly increased signal strength.

(62) Thus, in an automated fashion, an initial planar raster scan of the plinth surface is undertaken at the drop base centre, out to the drop edge. The software monitors and stores the Raman spectrum at each x, y position, and that with maximum intensity Raman signal is identified for the scan area.

(63) The obtained Raman spectra can be used individually, integrated or averaged over the full drop layer.

(64) The camera image is provided to a controller which sets the scan parameters, by extracting from the image the boundaries of the surface, to assist in ensuring that the scan covers the desired area. As an alternative, the camera could be omitted and the boundaries could be programmed into the system or detected by the detector (e.g. a sharp drop-off of signal at the edge of the droplet).

(65) FIG. 20 shows a schematic illustration of a three-dimensional scanning arrangement in which the same parts are denoted by the same reference numerals as in FIG. 19. In addition to motor controllers or manual controls that enable translation of the sample-receiving surface in the x-y plane as in FIG. 19, the sample-receiving surface may also be translated in the orthogonal z-direction. (Alternatively, the source or source optics could be translated to move the focal point 160 relative to the sample-receiving surface of the plinth 154.

(66) As indicated by the “slices” 162, the volume of the sample may be scanned completely by performing scans across the sample volume in planar regions separated by a small z-distance. Thus, a planar scan can be taken along the plinth surface, out to the edges of the drop. The procedure is repeated at fixed Z-offset distances, out to the upper edge of the drop; a z-axis motor offsets the z-axis position of the plinth (or of the source or source optics to translate the focal point to a new z-position), and the plinth is scanned in the next x-y plane a fixed distance offset in z, within the drop volume limits.

(67) The skilled person will appreciate that with x-, y- and z-controls, alternative scanning modalities are also enabled, such as scanning vertical slices (e.g. x-z plane or y-z plane) and then translating the plinth laterally to shift the plane where the slices are scanned, or in any other suitable scan pattern. Using the embodiment of FIG. 20, the apparatus may provide measurements from any point on the sample-receiving surface, or any point within the droplet volume bulk, or on the exposed droplet surface, as well as in the atmosphere surrounding the droplet. This can be used to determine areas of signal maximum, or to integrate the signal across the droplet, or to analyse the surface or surrounding atmosphere, depending on the analytical goal.

(68) Thus, the stored Raman spectra that have been taken at each point (or predetermined points assigned in software, for example, spectrum with maximum intensity Raman signal per scan layer) in each planar scan can be integrated or averaged into one Raman spectra for the drop volume.

(69) The camera is again optional but a preferred addition to the apparatus, in order to determine the extent of the droplet volume. Image recognition software can extract the position of the droplet surface and this can be translated to co-ordinates defining the boundaries of the scan (noting that the scan may extend outside the droplet in cases where analysis of gaseous components from the droplet are of interest). The use of a camera also enables the droplet volume to be calculated which may assist in other calculations, such as in determining refractive index, surface tension, and so on.

(70) FIG. 21 illustrates one embodiment for realising the scanning functionality described in relation to FIGS. 19 and 20. The embodiment of FIG. 21 is identical to that of FIG. 3 with the exception of the specifically identified changes now discussed. In place of the micrometer adjuster mechanisms 48 and 50 of the FIG. 3 embodiment, x-y adjustments are enabled by a first motorised actuator 170 allows for an x-direction translation of the flipping mechanism as a whole, a second motorised actuator 172 enables translation in the y-direction, and a third motorised actuator 174 enables translation in the z-direction. Each actuator is driven by a respective motor 176, with the motors under the common control of a motor controller (not shown) which may translate the entire cover mechanism and therefore the sample-supporting surface to any position within the limits of travel defined by the three actuators. Motor controller software may thus realise the scanning functionality described above, outputting appropriate control signals to the individual motors.

(71) The third motorised actuator may be omitted if z-scanning functionality is not required in simpler apparatuses. Alternatively, it may be provided on the optical components to scan the focal point of the incident light relative to the sample-supporting surface. However, it is preferred to include a z-motor on the optical components or the cover/sample-supporting surface in order to increase the utility of the apparatus, permit better focussing (or defocussing where desired) and enable volumetric scanning.

(72) The motors 176 in this embodiment (which is an example, and which the skilled person may vary according to design needs by using different motors and mechanisms) are stepper motors with 2045 steps per revolution, driving the actuators via M3×0.5 (ISO standard) threaded rods. Thus, a resolution of 244 nm per step is achieved. If such a resolution is not needed, then for example a stepper motor with 200 steps per revolution could be used, giving a resolution of 2.5 μm per step. The travel of each actuator is a minimum of 10 mm, so that a scan volume of 10 mm×10 mm×10 mm is achieved, enabling any part of a sample located in such a volume to be analysed.

(73) Each stepper motor is driven by a respective stepper motor PCB. A pair of micro switches (not shown) detect end position of travel of the cover assembly. A Raspberry Pi single board computer provides control signal outputs to the PCBs, according to programmed control instructions which may receive as inputs the image from the camera (if present) and from the detector. Thus, the program control may hunt for a signal maximum in a predefined scan pattern or it may perform a full-surface 2D scan or a full-volume 3D scan according to the needs of the user.

(74) The skilled person will readily appreciate that alternative control systems may be provided.

(75) As an alternative to stepper motors, linear motors could be employed, or piezometric inertial-slider (slip-stick) motor stages that can be actuated individually or together by application of a periodic exponential voltage to the piezoelectric elements on respective x-, y- and z-stages.

(76) An exemplary mechanism for enabling the cover assembly to be translated is illustrated in FIGS. 22 and 23. FIG. 22 shows the mechanism from above and FIG. 23 from below. In FIG. 22 there can be seen an outer frame 180 which is mounted to the housing of an apparatus (not shown). A first slider 182 is slidably mounted within the frame, and a second slider 184 is slidably mounted within the first slider 182. The flipping cover mechanism (not shown) is secured to the second slider 184, such that the sample-receiving surface locates within an aperture 186 in the second slider when the cover is in the closed position.

(77) Referring to FIG. 23, a first stepper motor 188 is mounted on the frame 180 and is connected to the first slider 182 so as to permit the controlled translation of the first slider relative to the frame. A second stepper motor 190 is mounted on the first slider 182 and is connected to the second slider 184 to permit the controlled translation of the second slider relative to the first slider. By appropriate control of the first and second stepper motors, the second slider 184 (and the flipping cover assembly mounted thereon) can be translated in the x- and y-directions relative to the frame (and hence the apparatus housing and the source and detector carried therein).

(78) While FIGS. 22 and 23 do not show a z-motor, the skilled person will appreciate that the mechanism of FIGS. 22 and 23 may be provided with a z-axis motor to enable the translation thereof in the z-direction. Alternatively, the source or appropriate optical components may be provided with z-axis controls in a straightforward manner to permit the relative translation of a focal point relative to the sample in the z-direction, and realisation of the volumetric scanning abilities discussed herein.

(79) In some cases, there will be an advantage in providing a mechanism to calibrate the movement of the x-y-z motors to ensure traceability of sample measurements. For example, in the pharmaceutical industry, “pill profiling” is important to the pharmaceutical industry. Many pills are formulated to include a coating, which may be added for a variety of reasons, such as taste masking, controlled or delayed release or dissolution, acid resistance in the stomach, and so on. For these reasons accurate control of coating thickness is important. Determining how the constituent ingredients of a pharmaceutical formulation are distributed, arranged in layers, blended, and so on, is a crucial part of the formulation process and of the analysis of formulations.

(80) The apparatuses of the invention enable samples to be analysed in a three-dimensional context, using the stepper motors to vary the point of analysis. However, for quality control purposes, it may be important to have the measurement position calibrated.

(81) Calibration can be enabled by providing a reference plinth manufactured using lithography e.g. from silicon with accurately manufactured features in the x-y plane for calibrating using the spectral maximum of the Raman signal from this substrate to calibrate the x-y motion of the stepper. Providing stepped features in this calibration plinth allows one to calibrate the z motion. There are numerous materials that could be used to fabricate such a plinth. Interferometrically polished quartz with evaporated Raman coatings are one example, though other methods of manufacture could be used to produce such a component to calibrate the stepper. The use of slip gauges could be developed to provide a quick calibration of the z-drive movement increasing confidence in measurements made using such apparatuses.

(82) FIG. 24 is a sectional schematic view of a further apparatus, employing an alternative mechanism to bring the sample in position to the flipping cover of earlier embodiments.

(83) The apparatus of FIG. 24 comprises a housing 200 shown in cut-away form, within which a spectrometer assembly 202 (which may be of any suitable type and configuration, including replaceable or built-in) is mounted. The spectrometer assembly is capable of translation within the housing from a first (lower) position to a second (higher) position as indicated by arrow 204. The assembly 202 houses a spectrometer having a source and a detector (not shown) and emits light 208 into an environmental chamber body member 210 mounted on top of the spectrometer assembly 202.

(84) A carrier 212 has a plinth removably mounted thereon to provide a sample-receiving surface 214, which is similar to previous embodiments. The carrier is rotatable on a spindle 216 about an axis passing through the carrier. The spindle is mounted at one end on a centre post 218 in the housing and on the other end by a roller 220 carried on a circular track 222 (albeit not appearing as truly circular in the drawing). Rotation of the carrier is achieved by a screw drive 224 and screw thread 226 at the top of the centre post 218. When the carrier is rotated the outer end travels around the circular track 222, and the carrier body itself spins about the axis of the spindle.

(85) A sample may be loaded on the carrier's sample-receiving surface 214, with the spectroscopic assembly 202 in the lowered position. The carrier may then be rotated until it is in position with the sample-receiving surface facing downward and positioned over the environmental chamber body member 210. The environmental chamber body member is then raised, and it makes a seal with the inverted carrier, so that the sample-receiving surface is enclosed and is exposed to the illumination from the source within the thus-formed environmental chamber.

(86) FIG. 25 is a perspective view of a sample holder (also called a drophead or plinth) 230, which has a circular drop-supporting surface divided into a major inner hydrophilic area 232 and a narrow annular hydrophobic band 234.

(87) An aqueous droplet 236 is positioned on the sample-receiving surface and is confined to the hydrophilic area. The droplet has an outer coating 238 of an oily composition which is immiscible with water and prevents evaporation, or may be used to study surface or interface phenomena and reactions. The boundary of the droplet is precisely pinned to the hydrophilic/hydrophobic boundary.

(88) Alternatively, the droplet coating 238 and droplet body 236 may be a partially immiscible combination; this enable the study of the dissolution and diffusion of the cap liquid into the droplet, and can provide valuable information about one or both liquids and their interaction.

(89) There exist commercial pipetting technologies and perhaps the simplest of these developments pipettes two phases of liquid. Such dual pipetting is useful for the analysis of medical fluids where these can be pipetted onto the plinth in one operation. The second immiscible liquid would produce a volume of sample trapped inside a cap of oil and this provides a sealed sample volume that does not evaporate and in which reaction kinetics can be studied in the bulk of the droplet, or at the oil-medial fluid interface. Such a sealed capped droplet sample could of course be produced by first pipetting a sample and then using a smaller pipette to deliver the oil, lipid solution or other hydrophobic liquid phase. It is possible of course to implement this embodiment as the converse with inner hydrophobic liquid on a hydrophobic area capped with a water solution that rests on a hydrophilic outer ring. There are a growing number of microfluidic systems that can be adapted to deliver such complex drop samples.