Infra-red spectroscopy system

Abstract

A sample slide (100) for use in a spectrometer (501), wherein the sample slide comprises a plurality of sample-receiving portions (111-114) provided on a sample side (115) of the slide, and a plurality of beam-receiving portions (121-124) provided on a beam-receiving side (125) of the slide, each beam-receiving portion being arranged opposite a respective sample-receiving portion, and wherein each beam-receiving portion is configured to act as an internal reflection element (IRE). A device (300) for use with a spectrometer (501) comprises a stage (330) configured to receive a sample slide (100); and a moving mechanism (360) configured to move the sample slide relative to a sample-measuring location (320) of the device. Associated methods for preparing a sample and measuring a sample are also disclosed.

Claims

1. A slide, comprising: a) a ample-receiving portion provided on a sample side of a slide; b) a beam-receiving portion provided on a beam side of said slide, said beam-receiving portion being arranged opposite said sample-receiving portion, wherein said beam-receiving portion comprises an internal reflection element (IRE); and c) a thickness between said beam-receiving portions and said opposite sample-receiving portion selected from the group consisting of 380 μm and 675 μm.

2. The slide according to claim 1, wherein said sample-receiving portion comprises a recessed portion surrounded by a raised portion.

3. The slide according to claim 1, wherein said sample-receiving portion is configured to receive or support a dry sample.

4. The slide according to claim 1, wherein said internal reflection element comprises adjacent grooves that are aligned or parallel and adjacent prisms that are aligned or parallel.

5. The slide according to claim 4, wherein each of said adjacent groove has a width in the range of 50-500 μm.

6. The slide according to claim 1, wherein each of said adjacent groove are spaced apart in the range of 0-200 μm.

7. The slide according to claim 1, wherein said slide further comprises silicon.

8. The slide according to claim 1, further comprising a slide holder, wherein said slide is on, within, or attached to said slide holder.

9. The slide of claim 1, wherein said slide is configured to interface with a FTIR spectrometer.

10. The slide of claim 1, wherein said slide is configured to interface with an ATR-FTIR spectrometer.

11. The slide of claim 1, wherein said internal reflection element comprises an infra-red transmissible material.

12. The slide of claim 11, wherein said infra-red transmissible material is selected from the group consisting of diamond, germanium, zinc selenide and silicon.

13. A method of preparing a sample for IR spectral analysis, the method comprising: drying one or more samples on a slide at a temperature of approximately 30-36° C. and/or under a gas flow rate of at least 50 m.sup.3/h, wherein said slide comprises: a) a sample-receiving portion provided on a sample side of said slide; b) a beam-receiving portion provided on a beam side of said slide, said beam-receiving portion, wherein said beam-receiving portion comprises an internal reflection element (IRE), and c) a thickness between said beam-receiving portion and said opposite sample-receiving portion selected from the group consisting of 380 μm and 675 μm.

14. The method of claim 13, wherein said temperature ranges between 34.5 to 35.5° C.

15. The method of claim 13, wherein said gas flow rate is at least 90 m.sup.3/h.

16. The method of claim 13, wherein said internal reflection element comprises an infra-red transmissible material.

17. The method of claim 16, wherein said infra-red transmissible material is selected from the group consisting of diamond, germanium, zinc selenide and silicon.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various aspects of the invention will now be described by way of example only, and with reference to the accompanying drawings, in which:

(2) FIG. 1 is a schematic representation of the principles of ATR-IR spectroscopy;

(3) FIG. 2 is a schematic representation of a conventional set-up showing a single IRE for performing ATR-IR spectroscopy analysis;

(4) FIG. 3 is a side view of a sample slide according to an embodiment of the present invention;

(5) FIG. 4 is a left hand side view of the sample slide of FIG. 3;

(6) FIG. 5 is a view from above of the sample slide of FIG. 3;

(7) FIG. 6 is a view from below of the sample slide of FIG. 3;

(8) FIG. 7 is an elevated perspective view of the sample slide of FIG. 3;

(9) FIG. 8 is an elevated perspective view of an alternative embodiment of a sample slide according to the present invention;

(10) FIG. 9 is a cross-sectional view of the sample slide of FIG. 3, showing a radiation beam reflected by the IRE;

(11) FIG. 10 is a cross-sectional view of the sample slide of FIG. 8, showing a radiation beam reflected by the IRE;

(12) FIG. 11 is an elevated perspective view showing an upper side of a holder holding the sample slide of FIG. 3;

(13) FIG. 12 is an elevated perspective view showing a lower side of a holder holding the sample slide of FIG. 3;

(14) FIG. 13 is an elevated perspective view showing a an upper side of a holder holding a sample slide according to another embodiment the present invention;

(15) FIG. 14 is an elevated perspective view showing a lower side of the holder of FIG. 13;

(16) FIG. 15 is an elevated perspective view showing a device for use with a spectrometer, according to an embodiment of the present invention;

(17) FIG. 16 is a view from above of the device of FIG. 16;

(18) FIG. 17 is a schematic cross-sectional view of an optical compartment of the device of FIG. 15;

(19) FIG. 18A is an ATR-FTIR spectrum showing investigation of an optimum angle of incidence for an IRE in the range of 30-80°;

(20) FIG. 18B is an ATR-FTIR spectrum showing investigation of an optimum angle of incidence for an IRE in the range of 30-50°;

(21) FIG. 18C is an ATR-FTIR spectrum showing investigation of an optimum angle of incidence for an IRE in the range of 30-50°, following spectral pre-processing;

(22) FIG. 19 is an ATR-FTIR spectrum showing the effect of the temperature used to dry a sample before analysis;

(23) FIG. 20 is an ATR-FTIR spectrum showing the effect of air flow applied to dry a sample before analysis;

(24) FIG. 21 is an ATR-FTIR spectrum showing the combined effect of temperature and air flow applied to dry a sample before analysis;

(25) FIG. 22 is a Table showing the mean area under a curve corresponding to the peak of absorbance for the hydroxyl group for different temperatures;

(26) FIG. 23 is a Table showing the mean intensity corresponding to the peak of absorbance for the amide I group for different temperatures;

(27) FIG. 24 is a Table showing the standard deviation for measurements corresponding to the peak of absorbance for the hydroxyl group for different temperatures;

(28) FIG. 25 is a Table showing the standard deviation for measurements corresponding to peak of absorbance for the amide I group for different temperatures;

(29) FIG. 26 is a schematic view of a method for analysing a sample according to an embodiment of the present invention;

(30) FIG. 27 is an elevated perspective view showing an upper side of a sample slide and holder according to another embodiment of the present invention;

(31) FIG. 28 is an elevated perspective view of a front side of a device for use with a spectrometer, according to another embodiment of the present invention;

(32) FIG. 29 is an elevated perspective view of a rear side the device of FIG. 28;

(33) FIG. 30 shows a conventional Specac Quest ATR-FTIR accessory; FIG. 31 shows the accessory of FIG. 30, fitted with a device according to an embodiment of the present invention;

(34) FIG. 32 shows the device of FIGS. 28-29, with the sample slide and holder of FIG. 27;

(35) FIG. 33 shows a comparison of spectral quality between three FTIR instruments using a sample slide of FIG. 3 (FIGS. 33(A)-(C)), and a gold-standard diamond ATR accessory (FIG. 33(D));

(36) FIG. 34 shows alternative approaches for SIRE integration in a FTIR spectrometer and relative SNR1 values, depicting; (a) the sample slide of FIG. 3 on an adapted ATR accessory; and (b) the sample slide of FIG. 3 on a specular reflectance accessory;

(37) FIG. 35 illustrates a principal component analysis (PCA) scatter plot of GBM (red) and non-cancer (blue) patients, with individual patients labelled so as to observe variability between SIRE wells;

(38) FIG. 36 is a cross-sectional view of an embodiment of a sample slide according to the present invention, showing certain dimension parameters;

(39) FIG. 37 shows the signal to noise ratio (SNR) of all 36 individual IRE designs investigated for various combinations of width, spacing and thickness;

(40) FIG. 38 shows a comparison of spectra obtained from designs of different thicknesses;

(41) FIG. 39 shows a comparison of peak intensities of IRE designs that have different distances between grooves;

(42) FIG. 40 shows a comparison between the spectra obtained from a multiwell silicon slide according to an embodiment of the present invention, and a conventional diamond IRE;

(43) FIG. 41 is a cross-sectional view of a specific embodiment of the sample slide of FIG. 36;

(44) FIGS. 42-44 illustrate a principal component analysis (PCA) scatter plot of three different patients undergoing treatment for melanoma; and

(45) FIGS. 45-49 illustrate the results of a spectral analysis carried out using a method according to an embodiment of the present invention, in respect of bacteria samples.

DETAILED DESCRIPTION

(46) Referring to FIG. 1 there is shown a schematic representation of the principles of ATR-IR spectroscopy. In FIG. 2, these principles are illustrated in the context of the conventional set-up showing a single IRE for performing ATR-IR spectroscopy analysis.

(47) As shown in FIGS. 1 and 2, “Attenuated Total Reflection” (ATR) employs an internal reflective element (IRE) 10 through which an IR beam 20 is passed. The sample 30 is deposited directly onto the IRE 10. The specific refractive properties of the IRE depends on the material from which the IRE is made, which can be for example diamond, germanium, zinc selenide or silicon. As shown in FIG. 1, when the IR light beam 20 is passed through the IRE 10 at an angle θ.sub.1 above the critical angle, the beam 20 is internally reflected through this medium on its upper surface 12 in contact with the sample 30. When the beam 20 meets the IRE and sample interface 12, this results in the production of an evanescent wave 14 which penetrates into the sample 30. The depth of this penetration is dependent upon the refractive indices of the IRE 10 and the sample 30, and is generally in the range of 0.5-2 μm. The beam 20, which then contains information about the sample 30, is then reflected by the IRE 10 towards a detector.

(48) FIGS. 3 to 6 illustrate various views of an embodiment of a sample slide, generally denoted 100, according to an embodiment of the present invention.

(49) As best shown in FIGS. 3 and 5, in this embodiment, the slide 100 has four sample-receiving portions 111, 112, 113, 114 provided on a sample side 115 of the slide 100 and arranged in a row. The provision of a sample slide 100 having multiple sample-receiving portions 111-114 allows the possibility of performing multiple measurements from a single slide 100 without having to remove and replace the sample slide 100 between successive measurements. This may avoid the need to remove, clean and dry an IRE between successive measurements as is current practice, thus permitting high throughput ATR-FTIR analysis.

(50) In this embodiment, the surface 116 of the slide 100 on its sample side 115 is substantially flat. However, in other embodiments, one or more of the sample-receiving portions 111-114 may define or may consist of a recessed portion, e.g. relative to an upper surface 116 of the slide 100. Alternatively, or additionally, one or more of the sample-receiving portions 111-114 may be surrounded by a raised portion, e.g. relative to an upper surface 116 of the slide 100.

(51) As best shown in FIG. 6, the sample slide 100 also has four beam-receiving portions 121,122,123,124 provided on a beam side 125 of the slide 100. Each beam-receiving portion 121-124 is arranged opposite a respective sample-receiving portion 111-114. Advantageously, each beam-receiving portion 121-124 is configured to act as an internal reflection element (IRE). For example, in this embodiment, one sample-receiving portion 121 may be used for background measurement, and three sample-receiving portions 122,123,124 to be used for triplicate measurements of a subject's sample, or for measurements of three separate samples from one or more subjects.

(52) Advantageously, the slide 100 contains and acts as the internal reflective elements (IRE(s)) required to perform ATR-FTIR analysis.

(53) As best shown in FIGS. 4 and 6, the beam-receiving portions 121-124 are configured to permit a radiation beam to penetrate a surface of the beam-receiving portions 121-124 on the beam side 125 of the slide 100.

(54) Each beam-receiving portion 121-124 defines has a plurality of elongate grooves 126 and prisms 127. Conveniently, each beam-receiving portion 121-124 defines has a plurality of aligned, parallel and adjacent grooves 126 and prisms 127.

(55) As best shown in FIGS. 4, 9 and 10, each groove 126 has a first groove face 128 and a second groove face 129. Each prism 127 also has a first prism face 128, corresponding to the first groove face, and a second prism face 129, corresponding to the second groove face 129.

(56) Each first groove face 128 or first prism face 128 is arranged to allow a radiation beam 20 to penetrate inwards a surface of a respective prism 127. Each second groove face 129 or second prism face 129 is arranged to allow a radiation beam 20 to penetrate outwards a surface of a respective prism 127.

(57) In the embodiment of FIGS. 7 and 9, the prisms 127 protrude outwardly relative to a lower surface 117 of the slide 100 on the beam side 125 thereof.

(58) In the embodiment of FIGS. 8 and 10, the prisms 127 are recessed relative to a lower surface 117 of the slide 100 on the beam side 125 thereof.

(59) Alternative embodiments may be envisaged in which an outer portion of the prisms 127 may protrude outwardly relative to a lower surface 117 of the slide 100 on the beam side 125 thereof, and an inner portion of the prisms 127 may be recessed relative to the lower surface 117.

(60) In the embodiments described herein, the thickness (t) of the slide 100 was manufactured to test a number of configurations and dimensions, and each type of slide was tested in the following thicknesses: 380 μm, 525 μm and 675 μm.

(61) Various dimensions of grooves 126 and prisms 127 were envisaged and experimented with, as illustrated in Table 1 below:

(62) TABLE-US-00001 TABLE 1 width (μm) depth (μm) spacing (μm) Design No. 100 70.6 25 1 50 2 100 3 150 105.9 25 4 50 5 100 6 200 141.2 25 7 50 8 100 9 250 176.5 25 10 50 11 100 12

(63) Further investigation regarding the configuration and design of the beam-receiving portions 121-124 acting as an IRE element for slide 100, was carried out, as represented by FIGS. 36-41.

(64) As shown in FIG. 36, each beam-receiving portions 121-124 acting as IRE elements has a thickness (t), and grooves 126 have a width (w) and are separated by spacing (s). Testing was performed for width (w) of 100 μm, 150 μm, 200 μm and 250 μm, spacing (s) of 25 μm, 50 μm and 100 μm, and thickness (t) of 380 μm, 525 μm and 675 μm. All slides were made of silicon.

(65) It was expected that a greater groove width (w) may allow more IR light to couple into the IRE elements (beam-receiving portions 121-124) and therefore improve signal throughput and intensity. It was also thought that decreasing the spacing between adjacent grooves (s) may reduce light scattering below the IRE well, reducing noise arising from the stray light recombining at the detector. Lastly, signal quality was expected to improve as the IRE thickness (t) decreased. This is because the effective path length of the IR beam through the IRE (beam-receiving portions 121-124) is reduced, thus preventing specific IR energy bands being absorbed into the material of the IRE itself which would result in a loss of signal at specific wavenumbers.

(66) SNR was calculated by taking the average signal value, x, of the Amide I band region (1625 cm.sup.−1 to 1675 cm.sup.−1) and dividing this by the standard deviation, σ, found across a region of the spectra where a high amount of noise can be found (1825 cm.sup.−1 to 1875 cm.sup.−1). Equation 1 expresses this:
SNR=x.sub.signal/σ.sub.noise  Equation (1)

(67) The results indicating signal-to-noise ratio (SNR) of all 36 individual IRE designs (representing each combination of width, spacing and thickness) are shown in FIG. 37. Colours indicate the 3 thicknesses investigated (red=380 μm, green=525 μm and blue=675 μm).

(68) It was concluded from FIG. 37 that a thickness of 380 μm would generate spectra with a more desirable SNR. This was an unexpected outcome as although the thickness of the IRE is expected to link to a higher information content below 1500 wavenumbers the significant improvement in signal-to-noise ratio demonstrates the superiority of the 380 micron approach.

(69) A comparison of spectra obtained from designs of different thicknesses is shown in FIG. 38. For this experiment, spacing (s) between grooves, and groove width (w) were kept constant in each case

(70) It can be se from FIG. 38 that the peaks of the 380 μm thick IREs are higher, indicating higher spectral intensity. It can also be seen that an improved signal intensity can be observed at the amide 1 and 2 band regions in thinner IREs. The improvement of thinner silicon IREs over thicker elements is further affirmed by way of a one-way ANOVA and Tukey post hoc comparison which confirms a significant difference exists between spectra obtained from 380 μm thick IREs and spectra obtained from 525 μm and 675 μm thick IREs (p<0.001 for a 95% confidence interval). Statistical analysis was carried out using Minitab.

(71) A comparison of peak intensities of IRE designs that have different distances between v-grooves is shown in FIG. 39 (green=25 μm, blue=50 μm and red=100 μm).

(72) This did not suggest a significant impact of groove width (w) and spacing (s) on SNR. There did not appear to be a discernible relationship between SNR and either groove width (w) or spacing (s) between grooves. However, when looking at the intensity of spectra where parameters width and spacing are kept constant, it appeared that a smaller spacing between grooves resulted in greater signal intensity (FIG. 39). This observation was consistent at all t and w values.

(73) Silicon naturally absorbs light of certain infrared frequency ranges. More specifically, silicon can cut-off signal below 1500 cm.sup.−1 wavenumbers as the beam is allowed to travel through the silicon for long enough. The effects of this can be reduced by decreasing the distance the beam travels through the silicon crystal. FIG. 40 shows a comparison between the spectra obtained from a multiwell silicon IRE of the present invention, and a conventional diamond IRE obtained from Specac Ltd.

(74) It can be seen from FIG. 40 that signals below 1500 cm.sup.−1 can still be acquired using the silicon IREs. However, effects of silicon lattice absorption can still be observed. At about 610 cm.sup.−1 wavenumbers a trough can be seen in the spectra acquired using Silicon IREs. This is to be expected, and is indicative of signal being lost as a result of internal lattice vibrations.

(75) The use of silicon as the material used to manufacture the slide 100 is particularly advantageous as this considerably reduces the costs associated with the manufacture of the slide 100, and allows the slide 100 to be used and marketed as a disposable slide, thus avoiding the need for cleaning and drying the slide before and/or after use.

(76) Based on the above observations, an advantageous embodiment of a slide according to the present invention was fabricated, illustrated in FIG. 41, having four 5×5 mm beam-receiving portions 121-124, and having a 380 μm thickness. The grooves were 250 μm wide, with a 25 μm spacing between them.

(77) Additionally, optimum angles for the first face 128 and second face 129 or the grooves 126 and prisms 127 were investigated, as explained in more detail with reference to FIG. 18. A suitable angle for a slide 100 made of silicon was found to be about 54.74° for a <100> silicon slide, and about 35.3° for a <110> silicon slide. It will be appreciated that the exact angle chosen for a give slide may depend on the material selected for manufacture of the slide, and/or on the expected angle of incidence of the irradiation beam.

(78) Referring now to FIGS. 9 and 10 there are shown cross-sectional views of the sample slide 100 of FIGS. 7 and 8, respectively, showing a radiation beam 20 being reflected by the IRE structure of the slide 100. It will be understood that the path of the beam is shown for illustration purposes, and that the actual beam path may depart from the path shown in FIGS. 9 and 10. For example, without wishing to be bound by theory, it is thought that, after the beam 20 has entered the slide 100, for example through a surface of first face 128 of a prism 127, the beam 20 may travel along a direction of the prisms 127, before being reflected on an internal surface 116 of the slide 100 and exiting the slide 100, for example through a second face 129 of another prism 127.

(79) Referring to FIGS. 11 and 12 there are shown an upper side and an underside of a holder 130 configured for holding the sample slide 100 of FIG. 3.

(80) The use of a slide holder 130 to hold the sample slide 100 may help prevent or reduce contact between a user and the sample slide 100, thus reducing contamination associated with handling the slide 100, which is particularly advantageous as the sample slide 100 comprises and acts as the internal reflective element(s). The use of a slide holder 130 also provides the sample slide with additional structural integrity, thus reducing the risk of damage or mechanical failure or the slide 100.

(81) Advantageously, the slide holder 130 has a size corresponding to the standard dimensions of a microscope slide, typically approximately 75×25×1 mm. This may help use of the sample slide 100 into conventional laboratories by conforming to existing handling procedures and avoiding the need to change common procedures.

(82) The sample holder has a sample side 136 and a beam side 137.

(83) On its sample side 136, the slide holder 130 has four windows 131-134, each window corresponding to and being of a similar size to a respective sample-receiving portion 111-114 of the slide 100. Typically, each window 131-134 and sample-receiving portion 111-114 has a size of approximately 5 mm×5 mm, which may allow each sample-receiving portion 111-114 to hold approximately 1-10 μL of sample, in use.

(84) On its beam side 137, the slide holder 130 has a window 135 having a size sufficient to expose the beam-receiving portions 121-124 of the slide 100. In an alternative embodiment, it may be envisaged that the side holder 130 may comprise or may define a plurality or windows on its beam side 137, each window or opening corresponding to a respective beam-receiving portion 121-124 of the slide 100.

(85) The slide holder 130 is provided with a tag region 138 suitable for receiving an identifier 139, which may be associated with the slide 100 received in the holder 130 and with the samples deposited on the slide 100.

(86) Another embodiment of a holder, denoted 130b, is shown in FIG. 27. The holder 130b of FIG. 27 is generally similar to the holder 130 of FIG. 11, like parts being denoted by like numerals, supplemented by the suffix “b”. The holder 130b of FIG. 27 has an arrow A showing the direction of movement of the holder 130b and slide 100b, in use, to align, sequentially, each of the beam-receiving portions (not shown) located opposite their respective sample-receiving portions 111b-114b with a beam of a spectrometer. The holder 130b also has markings 0,1,2,3 to allow easy labelling and referencing of each sample-receiving portion 111b-114b.

(87) Referring now to FIGS. 13 and 14 there are shown an upper side and an underside, respectively, of a holder 230 and corresponding sample slide 200, according to another embodiment the present invention. In this embodiment, the slide 200 has a 96-well plate configuration, that is, has 96 samples receiving portions 211 arranged in 8 rows of 12. Each well-receiving portion 211 has a corresponding beam-receiving portion 221 on a beam side 227 of the slide 200.

(88) On its sample side 236, the slide holder 230 has 96 windows 231, each window corresponding to and being of a similar size to a respective sample-receiving portion 211 of the slide 200.

(89) On its beam side 237, the slide holder 230 has a window 235 having a size sufficient to expose the beam-receiving portions 221 of the slide 200. In an alternative embodiment, it may be envisaged that the slide holder 230 may comprise or may define a plurality or windows on its beam side 237, each window or opening corresponding to a respective beam-receiving portion 221 of the slide 200.

(90) It will be appreciated that any number of sample-receiving portions may be envisaged for such grid-like or well-plate-like arrangement, depending on the number of measurements wishing to be made from a single slide 200.

(91) FIGS. 15 and 16 show an elevated perspective view and a top view, respectively, of a device, generally denoted 300, for use with a spectrometer, according to an embodiment of the present invention.

(92) The device 300 comprises an optical compartment 310 which has a plurality of optical elements 311-316 configured to guide a radiation beam 20 generated by the spectrometer to a sample-measuring location 320 (shown in FIG. 17) of the device 300.

(93) The device also has a stage 330 configured to receive a sample slide 340. In this embodiment, the sample slide 340 is a slide 100 as described with reference to FIGS. 3 to 12, and is provided within a holder 350 which is similar to the holder 130 described with reference to FIGS. 11 and 12. Thus, in this embodiment, the stage 330 is configured to receive and secure the slide holder 350 which holds the sample slide 340.

(94) The device 300 contains a moving mechanism 360 which is configured to move the sample slide 340 relative to the sample-measuring location 320. In this embodiment, since the sample slide 340 is provided within a slide holder 350, the moving mechanism 360 is configured to move the slide holder 350 which holds the sample slide 340, relative to the sample-measuring location 320.

(95) Because the slide 340 has four sample-receiving portions 341-344, the provision of a moving mechanism 360 arranged to move the sample slide 340 relative to the sample-measuring location 320 allows the analysis of multiple samples without having to remove and replace the sample slide 340 between successive measurements. This is advantageous when the sample slide comprises or includes one or more IREs, as this avoids the need to remove, clean and dry the IRE(s) between successive measurements.

(96) In this embodiment, the moving mechanism 360 is configured to move the stage 330. Since the sample slide 340 and slide holder 350 are stationary relative to the stage 330, moving the stage 330 causes the sample slide 340 to be moved relative to the sample-measuring location 320.

(97) In this embodiment, because the sample slide 340 has four sample-receiving portions 341-344 aligned in a longitudinal direction, the moving mechanism 360 is configured to provide unidirectional movement in the direction of alignment of the sample-receiving portions 341-344.

(98) However, if using a different slide, for example a slide 200 as described with reference to FIGS. 13 and 14, the moving mechanism 360 may be configured to provide bidirectional movement, for example along two perpendicular axes, in order to allow each of the 96 sample-receiving portions 211 to be sequentially aligned with the sample-measuring location 320.

(99) The moving mechanism 360 comprises a motor 362 for moving the stage 330. The moving mechanism 360, e.g. motor 362, can be controlled by an actuator (not shown), which can be activated manually and/or automatically.

(100) In use, the moving mechanism 360, e.g. motor 362, causes the stage 330, and therefore the sample slide 340, to move by a distance corresponding to the distance between two adjacent sample-receiving portions 341-344. By such provision, in use, the moving mechanism 360 allow the sample slide 340 to move sequentially, in order to align its sample receiving-portions 341-344 and beam-receiving portions with the radiation beam 20 in the sample-measuring location 320. This may allow automated and/or high throughput measurements of multiple samples using a conventional ATR-FTIR spectrometer.

(101) An embodiment of the optical compartment 310 of the device 300 is best shown in FIG. 17.

(102) The optical compartment 310 has a plurality of optical elements 311-316 configured to guide a radiation beam 20 generated by the spectrometer to a sample-measuring location 320 of the device 300. In this embodiment, the optical elements 311-316 are mirrors.

(103) The optical compartment 310 has walls that define an optical chamber 317. The optical compartment has an inlet 318 provided in a wall thereof, to allow the radiation beam 20 generated by the spectrometer to enter the optical compartment 310 and the optical camber thereof. The optical compartment has an outlet 319 provided in a wall thereof, e.g. a wall opposite the wall containing the inlet 318, to allow the radiation beam 20 reflected by the sample slide 340, to exit the optical compartment 310. The optical compartment 310 also has an opening (not shown) in an upper wall thereof to allow the reflected beam 20 to hit the sample slide 340 at the sample-measuring location 320.

(104) Typically, the inlet 318 and the outlet 319 are aligned with the normal direction of the radiation beam 20, i.e. aligned with the direction of the radiation beam 20 when the device 300 is not present. Thus, the device 300 may be considered to be accessory for use with a conventional spectrometer.

(105) Optical elements 311-313 are arranged to guide the radiation beam 20 to the sample-measuring location 320 at a predefined angle of incidence, and optical elements 314-316 are arranged to return the modified beam back towards the outlet 319. A person of ordinary skill in the art will appreciate that the optical elements 311-316, e.g. mirrors, may be arranged to guide or deliver the radiation beam 20 to the sample-measuring location 320 at a desired angle, which may depend on the configuration and material of the slide and IRE portion(s) thereof.

(106) Without wishing to be bound by theory, it is thought that an adequate angle of incidence for the radiation beam may be similar to or may be in the region of the angle of the the/a face 128,129 of the slide 100. For example in an embodiment with a <110> silicon slide having a face 128,129 angle of about 35.3°, the angle of incidence may be adjusted to be approximately 32°.

(107) One or more of the optical elements 311-316, e.g. each optical element 311-316, may be adjustable. By such provision, the angle of incidence of the radiation beam on the slide may be adjusted. This may allow the use of slides having different configurations and/or of slides made from different materials.

(108) FIGS. 28-29 show an elevated perspective view, from front and rear, respectively, of a device, generally denoted 300b, for use with a spectrometer, according to another embodiment of the present invention. The device 300b is generally similar to the device 300 of FIGS. 15-16, like parts being denoted by like numerals, but supplemented by the suffix “b”. However, in the embodiment of FIGS. 28-29, the device 300b does not have an optical compartment.

(109) The device 300b has a stage 330b configured to receive a sample slide 340b. The device 300b also has a moving mechanism 360b which is configured to move the sample slide 340b relative to a sample-measuring location 320b. In this embodiment, since the sample slide 340b is provided within a slide holder 350b, the moving mechanism 360b is configured to move the slide holder 350b which holds the sample slide 340b, relative to the sample-measuring location 320b. The device 300b has a switch 375b, and control buttons 371b-374b to control movement of the stage 330b and/or align a desired sample receiving-portions 341b-344b and beam-receiving portions with a radiation beam (not shown) at the sample-measuring location 320b.

(110) A view of the device 300b with a sample slide 100b and holder 130b is shown in FIG. 32. In this embodiment, the sample slide and holder are similar to the slide 100b and holder 130b as described with reference to FIG. 27.

(111) The stage 330b also has openings 331b-334b, each opening being configured to be substantially adjacent or aligned with a respective sample receiving-portion 341b-344b and beam-receiving portion of the sample slide 340b.

(112) It will be appreciated that, similarly to device 300 of FIGS. 15-16, device 300b may be configured for bi-directional movement of a sample slide with a plurality of wells arranged in rows, or movement of a plurality of sample slides in holders 100b and 130b.

(113) FIG. 30 shows a conventional Specac Quest ATR accessory 500 comprising an optical body 501 and a lid 502 which has a stage 503 to receive a sample on a conventional IRE and a compression arm 504 to maintain the IRE in position during analysis.

(114) FIG. 31 shows the accessory 500′ of FIG. 30, with its lid 502 removed, and replaced by the device 300b of FIG. 28-29. The device 300b is thus sized and configured to be connected to and fitted onto the spectrometer optical body 501. By such provision, the device 300b is configured such that, when the device 300b is installed or fitted on the spectrometer body 501, the sample measuring location of the device 300b is located at or substantially at a location where a sample would be placed using a conventional sample slide during use of the accessory 500′. Thus, the device 300b may be considered to be an accessory for use with a conventional spectrometer.

(115) A person of skill in the art will appreciate that other embodiments may be made which are sized and configured to fit other types of conventional spectrometers, such as, but not limited to as examples a Veemax optical accessory or those of a Perkin Elmer Spectrum Two or Thermo Fisher iS5 or Agilent Cary series, while using the same combination of a stage configured to receive a sample slide, and a moving mechanism configured to move the sample slide relative to a sample-measuring location.

(116) With reference to the FTIR spectra shown in FIGS. 18 to 21, these spectra contain two peaks of absorption that are of particular interest: the strong peak of absorption in the 3200-3500 cm.sup.−1 region which is characteristic of a water OH group, and the strong peak of absorption around 1690 cm.sup.−1 which is characteristic of a primary amide group (as found in proteins).

(117) Investigation of optimum angles of incidence are described with reference to FIGS. 18A-18C.

(118) FIG. 18A is an ATR-FTIR spectrum showing absorbance for different angles of incidence in the range of 30-80°. In FIG. 18B, the angles of incidence have been reduced to a range of 30-50°, as these angles were identified as providing optimum results. FIG. 18C is similar to the spectrum of FIG. 18B, but following spectral pre-processing, focussing on the absorbance region characteristic of primary amides. Spectral pre-processing typically involves a wavenumber selection approach to effectively ‘cut’ a fingerprint region of the spectrum, where typically the majority of biological molecules will present. Spectral pre-processing also typically includes a baseline correction (rubberband), to adjust for any scattering properties in the spectrum. Finally, and vector normalisation can be applied, which reduces the influence of sample inconsistencies, such as differences in thickness.

(119) In the spectra shown in FIGS. 19 to 21, protein-containing serum samples were analysed, under varying drying conditions.

(120) It has been discovered that, surprisingly, the drying conditions may affect the quality and reproducibility of the spectra obtained during subsequent analysis. In particular, it has been discovered that certain drying conditions may lead to improved reproducibility of analysis and/or sharpness in the spectra.

(121) FIG. 19 is an ATR-FTIR spectrum showing the effect of the temperature used to dry a sample before analysis. A 1 μL serum sample was deposited on a sample slide of the present invention and dried for 8 minutes at temperatures of T1=25° C., T2=30° C., and T3=35° C. It can be seen that drying the sample at 35° C. not only reduced the water content in the sample (smaller absorbance in the 3200-3500 cm.sup.−1 region), but also improved the absorption reading in the sample in relation to the primary amide group (absorption around 1690 cm.sup.−1), compared to samples dried for the same duration at lower temperatures.

(122) FIG. 20 is an ATR-FTIR spectrum showing the effect of air flow applied to dry a sample before analysis. A 1 μL serum sample was deposited on a sample slide of the present invention and dried for 8 minutes at room temperature of approximately 20° C. under air flows corresponding to a fan voltage of V1=5V, V2=12V, and V3=14V. It was measured that a fan voltage of 5V corresponded to a flow rate of approximately 15 m.sup.3/h, a fan voltage of 12V corresponded to a flow rate of approximately 99 m.sup.3/h, and a fan voltage of 14V corresponded to a flow rate of approximately 113 m.sup.3/h. It can be seen that drying the sample using a 14V fan voltage (hence under higher air flow) not only reduced the water content in the sample (smaller absorbance in the 3200-3500 cm.sup.−1 region), but also improved the absorption reading in the sample in relation to the primary amide group (absorption around 1690 cm.sup.−1), compared to samples dried for the same duration at lower fan voltages (hence under lower air flow).

(123) FIG. 21 is an ATR-FTIR spectrum showing the combined effect of temperature and air flow applied to dry a sample before analysis. A 1 μL serum sample was deposited on a sample slide of the present invention and dried for 8 minutes at 35° C. under air flows corresponding to a fan voltage of V1=5V, and V3=14V. It can be seen that, while a sample dried at 35° C. under low air flow (fan voltage of 5V) showed low moisture content and good primary amide absorbance peak, increasing the flow rate (fan voltage of 14V) further reduced the water content in the sample, and improved the sharpness of the primary amide peak of absorption.

(124) FIG. 22 is a Table showing the mean area (in arbitrary absorbance units.sup.2 (au.sup.2)) under a curve corresponding to the peak of absorbance for the hydroxyl group (absorbance in the 3200-3500 cm.sup.−1 region) for different temperatures and drying times. It is clear from FIG. 22 that increasing the drying temperature to 35° C. reduced the water content in the sample compared to samples dried at lower temperatures. It can also be seen that increasing fan voltage (and hence the air flow rate) also reduced the water content in the sample compared to samples dried at lower fan voltages. Finally, it can be seen that the combined effect of a drying temperature of 35° C. with a high air flow (12V or 14V fan voltages) produced the best results. In particular, the drying time required to dry the sample was significantly reduced at 35° C. compared to lower temperatures. For example, a value of less than 12 was achieved after a drying time of 2 minutes under a temperature of 35° C. and with fan voltage of 5V, 12V or 14V, whereas, at 30° C., the water content was higher even after 6 minutes drying time.

(125) FIG. 23 is a Table showing the mean intensity corresponding to the peak of absorbance for the amide I group for different temperatures. It can be observed that increasing the temperature and air flow each led to increased absorbance intensity characteristic of the amide I group. In other words, there was a clear correlation between the dryness of the sample (low water content as observed in FIG. 22) and the measured intensity of the amide I peak absorbance in the sample, which was unexpected.

(126) FIG. 24 is a Table showing the standard deviation for measurements corresponding to the peak of absorbance for the hydroxyl group for different temperatures. FIG. 25 is a Table showing the standard deviation for measurements corresponding to peak of absorbance for the amide I group for different temperatures.

(127) The standard deviation is representative of the reproducibility, with a lower standard deviation meaning better reproducibility. It can be seen from FIGS. 24 and 25 that drying the sample at higher temperature (35° C.) and under air flow each improved reproducibility of the ATR-FTIR analysis, with the best results being achieved for a combination of a drying temperature of 35° C. with application of air flow.

(128) FIG. 26 is a schematic view of a method 400 for analysing a sample according to an embodiment of the present invention.

(129) Step 410 illustrates the step of collecting a serum sample 415. Whole blood was drawn from a patient. Following this the blood underwent a centrifugation step to isolate the blood serum other blood components. Typically, a minimum of 5 ml of serum is required per patient. Samples were snap-frozen for storage and thawed to room temperature for analysis.

(130) Step 420 illustrates the step of dispensing a serum sample 415 on onto a sample slide 430. The sample slide 430 was a slide according to an embodiment of the present invention as described with reference to FIGS. 3-7 and 11-12.

(131) 5 μL of a samples were pipetted into wells 432, 433 and 434, while well 431 was left empty, to be used as background control.

(132) Step 440 illustrates the step of drying a batch of slides 430a, 430b, 430c. The slides were stacked and placed in a drying unit 442 for drying. The drying unit 442 was set to a temperature of about 35° C. and the slides were allowed to dry for about 2 minutes using a 5V fan to provide an air flow rate of about 15 m.sup.3/h.

(133) Step 450 illustrates the step of performing spectral analysis of the samples.

(134) The instrument used in the analysis of the samples is a Spectrum 2™ FTIR spectrometer from Perkin Elmer. This spectrometer is fitted with an accessory device 300 as described in the embodiment of the present invention referring to FIGS. 15-17. This facilitates high-throughput analysis of samples. The slide 430 was provided with an identifier 439 fixed on one end of the slide 430 to easily and reliably identify the original of the samples. In this embodiment, the closest well 431 to the identifier 439 was used as a ‘background’ well 431. Typically, the slides 430 were placed on the stage 330 of the device 300 such that the background well 431 is analysed first by the spectrometer. A person of skill in the art will appreciate that the purpose of a blank well is to serve as a background scan of the environment for the spectrometer instrument. This collects all spectral information from the environment and removes it from the data collected from the subsequent serum samples. Therefore, a background measurement is typically carried out before analysis of samples to be analysed. This ensures that, in the context of analysing serum samples, important information from the serum is not obscured by components in the surrounding environment.

(135) As described with reference to FIGS. 15-17, once ATR-FTIR measurement is complete for first well 431, the apparatus 300 moves the slide 430 relative to the sample-measuring location 320, for the sample in the second well 432 to be analysed. Thus, each sample in wells 431,432,433,434 is analysed by ATR-FTIR spectrometry without the need to remove the slide 430 between measurements.

(136) The spectrometer was configured in the following manner: a resolution of 4 cm.sup.−1, a 4 cm.sup.−1 aperture, and with 32 scans per sample and background. This is a standard ATR-FTIR spectrometer setting which allows spectra to be taken typically in under a minute.

(137) Step 460 illustrates the step of processing the data and presenting the information to a user via a user interface 462. The present method allows results to be delivered in real time and presented to the user as with a simple interface 462 displaying the result with the percentage level of confidence.

(138) Once analysis has been completed, the slide 430, which also acts as the IRE for each well 431,432,433,434, may be disposed of appropriately, as will typically be treated as biological waste for disposal. Alternatively, the slide 430 may be stored for future reference, or cleaned and re-used as appropriate to the application.

(139) Experimental Data

(140) Use of Sample Slide with Conventional Spectrometers (FIG. 33)

(141) A sample slide according to an embodiment of the present invention was prepared, consistent with the embodiment of FIGS. 3-6. The slide was made of silicon.

(142) A sample was prepared with human pooled serum, and applied to each of the three sample wells, the first well being used for baseline reference. The same sample slide was used throughout so as to reduce any differences arising from sample preparation. A single spectrum was obtained from each sample well and recorded. Spectra were then pre-processed using a rubberband baseline correction followed by vector normalisation. These steps aim to negate any background effects arising from unwanted light scattering, as well as sample variations such as thickness which can have multiplicative effects on the spectra. Pre-processing thus makes all spectra comparable.

(143) To compare data regarding spectral quality, and thus system performance, values of signal-to-noise (SNR) were extracted. A higher SNR can be regarded as preferable as important spectral information is larger in comparison to the unwanted background noise. SNR can be calculated by comparing intensity values of a ‘signal’ region against a ‘noise’ region. The amide I peak of a biological spectrum is often the most intense due to fundamental vibrations from protein and thus the maximum absorbance value of this peak is often used as a signal region. Noise values can be obtained from anywhere in the IR spectrum that is free of vibrational modes found in biological samples and modes arising from ambient conditions. The regions between 4000-3700 cm.sup.−1, 2800-2500 cm.sup.−1, 2000-1800 cm.sup.−1 and below 900 cm.sup.−1 are commonly chosen noise regions. In this instance, maximum absorbance values between 1900-1850 cm.sup.−1 and 900-850 cm.sup.−1 were selected as two separate measures of signal quality. The former area was chosen to avoid contributions from the water overtone region; the latter, to encompass loss of sensitivity in lower wavenumbers due to the use of silicon, whilst also addressing limitations in detector sensitivity.

(144) FIG. 33 shows a comparison of spectral quality between three FTIR instruments using a sample slide of FIG. 3 (FIGS. 33(A)-(C)), and a gold-standard diamond ATR accessory (FIG. 33(D), and in particular: (A) Perkin Elmer Spectrum 2 FTIR, (B) Thermo Fisher Nicolet is 5 FTIR, (C) Agilent Technologies Cary 670 FTIR, and (D) Perkin Elmer Spectrum 2 with uATR accessory (gold standard).

(145) SNR values are given for each set of spectra, with the amide I peak absorbance compare to the maximum absorbance value in the 1900-1850 cm.sup.−1 region (SNR1), and the maximum absorbance in the 900-850 cm.sup.−1 region (SNR2)

(146) It can be seen from FIG. 33(D) that the gold standard approach of using a commercially available ATR accessory with integrated diamond crystal yields high quality spectra with high SNR. FIGS. 33(A)-(C) show that using the samples slides according to an embodiment of the present invention, integrated onto a universal ATR accessory (using a Slide Indexing unit), in conjunction with three other commercially available spectrometers, the Perkin Elmer Spectrum 2 FTIR spectrometer produces spectra with equivalent quality to the gold standard approach (FIG. 33(A)). Good quality spectra are also acquired from Thermo Fisher Nicolet iS 5 (FIG. 33(B)) and Agilent Cary 670 (FIG. 33(C)) systems.

(147) Comparison of Silicon IREs (‘SIREs’) (FIG. 34)

(148) Alternative approaches to the approach described in relation to FIG. 33 were explored using adapted IRE interfaces with commercially available ATR (FIG. 34(a)) and specular reflectance accessories (FIG. 34(b)). As shown in FIG. 34, the SNR for each approach is distinctly lower than the spectral quality obtained from a gold-standard approach such as a diamond IRE system, and considerably less than the system described above and investigated in relation to FIG. 33.

(149) Clinical Classification Study on SIREs (FIG. 35)

(150) The diagnostic performance of ATR-FTIR has been established using proof-of-concept studies using diamond IRE based ATR. These retrospective studies determined that brain tumour patients could be distinguished at sensitivities and specificities of 92.8% and 91.5% respectively. In order to investigate the diagnostic performance of a SIRE-based approach, a small classification study was conducted on 15 glioblastoma multiforme (GBM) and 15 control patients. A small study such as this would provide an indication of potential performance of this new approach. One important consideration with a small dataset is that some computational methods would not be suitable, due to the risk of overfitting and insufficient validation.

(151) For all patients, 3 μL of serum was pipetted onto each of the wells ‘1’, ‘2’ and ‘3’ on a sample slide according to the embodiment of FIG. 3, and allowed to dry at room temperature (20-22° C.). Three spectra were obtained per well resulting in 9 spectra per patient, and an overall total of 270 spectra. Spectra were pre-processed by cutting to the fingerprint region (900-1000 cm.sup.−1), second-order differentiated and vector normalised. As the number of spectra is less than the number of variables in the dataset, supervised analysis was deemed unsuitable and as such multivariate analysis, in this case principal component analysis (PCA), was conducted to observe differences between cancer and non-cancer patients (FIG. 35).

(152) PCA is a technique used to emphasize variation and highlight patterns in a dataset. In the present case, the PCA transformation was carried out in 2 dimensions, with the first principal component (PC1) being associated with the wavenumber and absorbance showing the largest variance in the dataset, and the second principal component (PC2) being associated with the wavenumber and absorbance showing the second largest variance in the dataset. In other words, PCA analysis reduces a spectrum into variables that account for variance within the dataset. As such, when these variables are compared against each other in a scatterplot, separation between classes in the axes suggests biological variation. Conversely, relative closeness or overlap suggests biological similarity.

(153) If cancer and non-cancer appear to separate in a PCA scatter plot, this suggest that the two classes are distinguishable using the FTIR approach. What can be seen in FIG. 35 is that there is a distinct split between cancer and non-cancer patients, with very little overlap. This is promising for any subsequent analysis, such as the use of classification algorithms that would extract sensitivity and specificity, as an unsupervised approach is able to initially identify spectral differences indicative of disease status.

(154) A further clinical classification study was carried out for patients suffering from melanoma. The approach taken was similar to the above study as illustrated in FIG. 35, but with regard melanoma-type cancers rather than brain tumors.

(155) FIGS. 42-44 shows the results of a principal component analysis (PCA) carried out for three different patients being monitored during the course of their treatment. The sample analysis in each case was carried out as described in the method 400 of FIG. 26, followed by PCA analysis.

(156) With reference to FIG. 42, samples taken from five different patient visits were analysed. The patient had melanoma in visit 1, and a relapsed occurred at visit 3. As shown in FIG. 42, PCA analysis of the results showed that the samples associated with visit 2 were distinctively different and separated from the other samples. The results shown on FIG. 42 clearly show that the present method allow identification of the presence or absence of cancer (in this case melanoma) in a patient's biological sample.

(157) Similarly, with reference to FIG. 43, the patient had melanoma in visit 1, and a relapsed occurred at visit 3. Again, PCA analysis of the results showed that the samples associated with visit 2 (no melanoma) were distinctively different and separated from the other samples (melanoma).

(158) The patient associated with FIG. 44 was different in the sense that the patient had no melanoma in visits 1, 3 and 4, but a relapsed occurred at visit 2. Thus, in this case, the samples which were distinctively different and separated from the other samples were the samples which identified the presence melanoma.

(159) FIGS. 45-49 illustrate the results of spectral analyses carried out using a method according to an embodiment of the present invention, in respect of bacteria samples.

(160) FIG. 45 shows average spectral data highlighting spectral differences in the six different bacteria families that the 86 samples that were analysed belong to.

(161) FIG. 46 shows average spectral data highlighting spectral differences in bacteria according to bacteria genus.

(162) FIGS. 47-48 show PCA analysis PC1 vs PC2, and PC1 vs PC3, respectively, highlighting the differences between bacteria families.

(163) FIG. 49 illustrates the PC loadings spectral discrimination associated with the PCA analysis of FIGS. 47-48. Separation between bacterial families is shown via scatter plots and corresponding loadings show that this is due to distinctive spectral regions.

(164) Thus, it can be see that the methods and systems of the present invention are not limited to the investigation of cancerous samples, but may also be applied to numerous other applications, including for example the investigation and identification of different types of bacteria families of genus. Further, it will be appreciated that the methods and systems of the present invention are not limited to the investigation of biological samples, but may also be applied to measuring or testing non-biological applications, for which spectrometry, e.g. ATR_FTIR spectrometry, may lead to useful results interpretation and/or classification.

(165) It will be appreciated that the described embodiments are not meant to limit the scope of the present invention, and the present invention may be implemented using variations of the described examples.