PATENT DOCUMENT

Abstract:
It has been discovered that specially structured metallic films containing voids can deliver a hugely enhanced surface enhanced Raman spectroscopy (SERS) effect. By selecting a particular size and geometry for the voids, metallic films can be provided which have an enhanced photon-to-plasmon conversion efficiency for incident radiation of a predetermined wavelength. Controllable surface-enhanced absorption and emission characteristics may thus be provided, which are useful for SERS and potentially also other optical spectrometry and filtering applications. With such a large Raman signal, the invention enables fast, compact and inexpensive Raman spectrometers to be provided opening up many new application possibilities.

Full Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates principally but not exclusively to Raman spectroscopy, in particular surface enhanced Raman spectroscopy (SERS). 
       BACKGROUND OF THE INVENTION 
       [0002]    Raman spectroscopy is used for a variety of applications, most commonly to study vibrational quanta, such as vibrations in molecules or phonons in solids, although other quantised entities can also be studied. Raman spectroscopy can provide detailed information relating to the physical state of sample materials and can be used to distinguish various states of otherwise chemically identical molecules, such as various molecular isomers, from one another. 
         [0003]    Raman spectroscopy finds wide ranging use in numerous different industries. By way of example, Raman spectroscopy finds application in the pharmaceutical, chemical, bio-analysis, medical, materials science, art restoration, polymer, semiconductor, gemology, forensic, research, military, sensing and environmental monitoring fields. 
         [0004]    Although Raman spectroscopy is an extremely useful analytical tool, it does suffer from a number of disadvantages. The principal drawbacks associated with Raman spectroscopy arise because of the small scattering cross-section. Typically, only 10 −7  of the photons incident on the sample material will undergo Raman scattering. Hence, in order to detect Raman scattered photons, Raman spectrometers typically employ high power laser sources and high sensitivity detectors. Not only is the scattering cross-section small in an absolute sense, but it is small relative to Rayleigh scattering in which the scattered photon is of the same energy as the incident photon. This means that there are often problems related to separating out the small Raman signal from the large Rayleigh signal and the incident signal, especially when the Raman signal is close in energy to the incident signal. 
         [0005]    High power sources are not only both bulky and expensive, but at very high power the intensity of the optical radiation itself can destroy the sample material, thus placing an upper limit on the optical radiation source intensity. Similarly, high sensitivity detectors are often bulky and expensive, and even more so where forced cooling, such as with liquid nitrogen, is necessary. Additionally, detection is often a slow process as long integration periods are required to obtain a Raman spectrum signal having an acceptable signal-to-noise ratio (SNR). 
         [0006]    The problems associated with Raman spectrometry have been known long since C. V. Raman discovered the effect itself in 1928. Since that date, various techniques have been applied to improve the operation of Raman spectrometers. 
         [0007]    Certain of the techniques involve the use of metal surfaces to induce surface plasmon resonance (SPR) for more efficient coupling of energy into the sample material. One refinement of this technique involves placing sample material on or near a roughened surface. Such a surface can be formed by the deposition of metallic/dielectric particles, sometimes deposited in clusters [1-3]. The roughened surface is found to give rise to an enhanced Raman signal, and the technique of using the roughened surface to obtain a Raman spectrum is known as surface enhanced Raman spectroscopy (SERS). 
         [0008]    However, whilst SERS devices can lead to an improved SNR when compared to previous conventional Raman spectrometers, they still suffer to a lesser extent with various of the same disadvantages. For example, SERS devices are still not efficient enough to provide a Raman signal without fairly long detector integration times, and can still require the use of bulky and expensive detectors. Even at present, an acquisition time for a Raman spectrum of some five seconds is considered to be extremely good. 
       SUMMARY OF THE INVENTION 
       [0009]    According to a first aspect of the invention, there is provided a spectrometer for obtaining a Raman spectrum from a sample material. The spectrometer comprises an optical source for generating optical radiation, a substrate for receiving the optical radiation, and a spectral analyser for analysing the Raman scattered radiation emerging from the substrate. The substrate comprises a metallic film that incorporates a plurality of voids of a predetermined size. These voids are suitable for confining surface plasmons. The surface plasmons couple energy from incident optical radiation to a sample material when the sample material is located proximal the substrate. The surface plasmons are also responsible for converting scattered energy emitted from the sample material into the Raman scattered radiation. The substrate may be incorporated into existing Raman spectrometers in order to improve their performance. 
         [0010]    The substrate provides an enhanced Raman signal. Therefore to obtain an acceptable SNR, less incident optical radiation or a lower sensitivity detector can be used, or both. In various embodiments, the spectral analyser makes use of detectors that do not need to be cooled, such as a photodiode array. Certain embodiments can make use of high efficiency compact optical source devices, such as a laser diode or laser diode array. By employing such detectors and arrays, a high efficiency, low-power, portable, and compact Raman spectrometer can be provided. Moreover, embodiments employing, for example, a laser diode array provide optical radiation that can be used to illuminate large area of substrate. In various such embodiments, it is not always necessary to focus the optical radiation, thereby further improving the compactness and reducing the cost of these spectrometer. 
         [0011]    Moreover, because of the enhanced Raman signal, input channel optics provided with the spectral analyser to collect Raman scattered radiation may be made to differ from optics used in conventional Raman spectrometers. In particular, various embodiments avoid the need to use a high numerical aperture lens system to collect Raman scattered radiation. This allows the collection optics to be spaced away from the substrate. Such spacing is particularly beneficial as it enables fluids containing sample material to be analysed to freely flow over the substrate without being impeded by the collection optics. The fluid may be liquid or gas. The input channel optics may comprise a fibre optic input channel oriented towards the substrate. As in various embodiments the direction of emerging Raman signal can be predicted, use of a fibre optic input channel can be used to cut down on any background signal from the optical source that reaches the spectral analyser. 
         [0012]    Further, since the signal is strong, alignment and focusing tolerances of the optical components are much relaxed so that, for example, the need to provide for adjustability of the optical components to allow signal optimisation before each experimental series can in some cases be dispensed with entirely. 
         [0013]    Furthermore, since the Raman signal is enhanced, a Raman spectrometer incorporating the substrate is able to acquire a Raman spectrum having an acceptable SNR using a reduced integration time. Not only does this enable faster processing of sample materials, but it also opens up the exciting possibility of using Raman spectroscopy to monitor processes in real-time, such as chemical reactions and catalysis processes. 
         [0014]    Although the applicants have for several years been involved in research relating to producing and investigating the optical properties of metallic films which include voids [4, 5, 6, 9], the fact that these films were capable of delivering huge SERS enhancement was not previously realised since their physical structure differs greatly from that of any surfaces previously used. 
         [0015]    However, when it was tried out, the results showed huge enhancements in the Raman signals. These initial experiments indicated that the Raman signal could be increased by a factor of between some 10 4  to 10 14  when compared to non-SERS apparatus. 
         [0016]    Moreover, experimental and theoretical investigations detailed below have indicated that the Raman signal can be increased by at least a factor of two when compared to conventional SERS apparatus by careful design of the voids to optimise them for particular wavelengths of incident optical energy. 
         [0017]    Additionally, the theoretical and experimental studies show that by careful design of the voids, the Raman signal can be concentrated to be emitted at a predetermined angular direction, thereby allowing appropriately positioned low NA collection optics to be used to collect the signal. 
         [0018]    Whilst the origins of the enhanced Raman signal are not completely understood, it is believed that it may be due to the effect of localised plasmons that form at the surfaces of the voids. It is thought that the localised plasmons increase the coupling efficiency between the incident optical radiation and any sample material located proximal the surface of the metallic film, and subsequently give rise to the dramatic enhancements in Raman signal strength that are seen by the applicant. 
         [0019]    In various embodiments, the voids have the shape of a truncated sphere. By controlling the diameter of the sphere and the thickness of the truncation, the emission direction of a particular wavelength of Raman signal can be tailored in a known and predictable manner detailed further below. Additionally, by providing part-spherical voids with truncation parallel to a surface of the substrate, the emission direction remains predictable and constant even if the substrate is rotated about an axis normal to the surface. Alignment of the substrate within the spectrometer is thereby facilitated. 
         [0020]    The size of the voids may be selected depending upon the wavelength of the optical radiation that is to be used with a particular sample material. Substrate responses may thus be tailored to suit a particular sample material. The voids may range from about a few nanometres to about many tens of microns in size. For example, the size of the voids may range from about 10 nm to 50 nm for working with deep ultraviolet radiation, to about tens of microns for working with mid-infrared radiation tuned to be resonant to molecular vibrational transitions. In other examples, a void may be provided with a diameter from about 100 nm to about 900 nm due to the ease of manufacturing voids of this size. For still further examples, the size of the voids may correspond substantially to the wavelength of visible optical radiation. Voids may be used with optical radiation that is selected so to be non-ionising and so as not to induce extraneous molecular vibrations for a particular sample material. This allows the optical radiation merely to probe the sample material without unduly influencing it. 
         [0021]    Certain embodiments include a substrate that is generally planar in shape and in which the voids are uniformly spaced over at least part of a planar surface of the substrate. Efficient use can thus be made of the surface, and a uniform signal for Raman spectra can be obtained from different parts of the substrate surface. 
         [0022]    Various embodiments incorporate a substrate that further comprises a waveguide structure for coupling the optical radiation to a sample material through the metallic film. Where such a waveguide structure is provided, the spectral analyser may also be configured to collect Raman scattered radiation that emerges from the waveguide. 
         [0023]    According to a second aspect of the invention, there is provided a method of obtaining a Raman spectrum from sample material. The method comprises introducing sample material into the spectrometer according to the first aspect of the invention proximal to the substrate, activating the optical source and operating the spectral analyser to provide the Raman spectrum of the sample material. 
         [0024]    The method may comprise a step of introducing sample material by flowing a fluid containing the sample material across the substrate in a region illuminated by the optical radiation. The substrate is particularly good for this because, besides being positionable away from any light collecting optics, it may be provided with a smooth surface. 
         [0025]    In various embodiments, the method comprises varying the electric potential of the metallic film of the substrate. Applying a electric potential to the metallic film allows the dynamics of the sample material proximal the surface of the voids to be monitored. Moreover, it can permit real-time surface reaction monitoring, enable chemical reactions to be initiated, enable the breakdown of various molecules to be monitored, and be used to provide information about how Raman spectra are modified by the presence of electric fields. 
         [0026]    According to a third aspect of the invention, there is provided a method of making a substrate having an enhanced efficiency of coupling optical energy to surface plasmons at a predetermined wavelength of optical radiation incident upon the substrate. The method comprises determining the size and shape of voids which when formed in a metallic film efficiently couple optical energy at the predetermined wavelength to surface plasmons that form in the voids, and forming a substrate comprising a metallic film that includes a plurality of voids of the determined size and shape. 
         [0027]    The size and shape of the voids determines whether optical radiation of a particular predetermined energy will couple into plasmons that form at the surface of the voids. Furthermore, the applicant has found that by modifying the size and shape of the voids and the incident direction of the optical radiation, both optical-to-plasmon and plasmon-to-optical energy couplings can be controlled as well as the orientation of optical radiation emitted from the metallic film. 
         [0028]    Voids may be formed in the metallic film that are uniformly spaced over a surface of the substrate. A waveguide structure may be formed in the substrate for coupling optical radiation from the substrate through the metallic film. 
         [0029]    In various embodiments, the voids are in the shape of a truncated spherical void. The size of these voids are determined in dependence upon the desired wavelength of the optical radiation. The diameter of the truncated spherical void may be chosen to be of the same order of magnitude as the predetermined wavelength of optical radiation. For example, the diameter of the truncated spherical void may be chosen to be about equal to the wavelength of optical radiation. In various examples, the diameter of the truncated sphere is from about 50 nm to about 10,000 nm, or about 100 nm to about 900 nm. The thickness of the truncated spherical void may be chosen to couple optical energy at the predetermined wavelength to zero-dimensional plasmons that form in the void. 
         [0030]    The substrate may be formed by depositing a template of ordered spherical particles on a substrate surface, and passing a predetermined amount of charge though a metallic ion containing solution that surrounds the template so as to deposit the metallic film on the substrate surface. 
         [0031]    The third aspect of the invention relates to how to apply the experimental and theoretical information obtained by the applicant so as to design and manufacture substrates having tailored emission characteristics. Through the applicant&#39;s investigations, the applicant has come to understand how to produce the metallic films necessary for efficient use in various applications or with various sample materials. Numerous applications for such substrates are envisaged. For example, applications are envisaged in spectrometry, such as Raman spectrometry, and in optical filtering. 
         [0032]    According to a fourth aspect of the invention, there is provided a substrate made according to the method of the third aspect of the invention. Such substrates may incorporate a metallic film that comprises one or more of the following materials: gold, platinum, silver, copper, palladium, cobalt and nickel. It will be appreciated that the metallic film may be made of any one of these elements alone or in combination with each other or other materials to form an alloy. Materials that have catalytic properties, inert properties, optically beneficial properties, etc. may be preferred depending upon the application of the substrate. For example, silver may be used to provide a high Raman enhancement signal in applications where it is unlikely to be placed in contact with oxidising materials that would otherwise degrade its optical performance. The substrates may be encapsulated. 
         [0033]    In various embodiments the substrate may be provided already with a sample material for analysis provided in the voids of the metallic film. In certain embodiments, the sample material is an organic material. Provision of substrates with sample materials is convenient for users, particularly where the sample materials have undesirable chemical or biological properties, such as high toxicity. 
         [0034]    According to a fifth aspect of the invention, there is provided an optical device incorporating the substrate according to the fourth aspect of the invention. A sixth aspect of the invention relates to the use of the optical device according to the fifth aspect of the invention. For example, such an optical device may be a filter device, an analysis device or a device other than a Raman spectrometer. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0035]    For a better understanding of the invention and to show how the same may be carried into effect reference is now made by way of example to the accompanying drawings in which: 
           [0036]      FIG. 1  shows a conventional Raman spectrometer; 
           [0037]      FIG. 2  shows a first embodiment of a Raman spectrometer according to the present invention; 
           [0038]      FIG. 3  shows a second embodiment of a Raman spectrometer according to the present invention; 
           [0039]      FIG. 4  shows a third embodiment of a Raman spectrometer according to the present invention; 
           [0040]      FIG. 5  is a flow diagram illustrating a method of obtaining a Raman spectrum from a sample material according to an embodiment of the invention; 
           [0041]      FIG. 6  shows a Raman spectrum of benzenethiol obtained using the first embodiment of a Raman spectrometer according to the present invention; 
           [0042]      FIG. 7  shows a set of Raman spectra of pyridine obtained with different electric potentials applied in solution to the metallic film of the first embodiment of a Raman spectrometer according to the present invention; 
           [0043]      FIG. 8  shows modelled data indicating predicted Raman signal enhancement factors for substrates incorporating metallic films made using various metals in accordance with the present invention; 
           [0044]      FIG. 9  is a flow diagram illustrating a method of making a substrate having an enhanced efficiency of coupling optical energy to surface plasmons at a predetermined wavelength of optical radiation according to an embodiment of the present invention; 
           [0045]      FIG. 10A  shows a schematic illustration of a plasmon formed in a void according to various embodiments of the present invention; 
           [0046]      FIG. 10B  shows a schematic illustration of a void having a truncated spherical shape for use in various embodiments of the present invention; 
           [0047]      FIG. 10C  shows a perspective view of a metallic film in a substrate according to an embodiment of the present invention; 
           [0048]      FIG. 10D  shows a plan view of the metallic film of  FIG. 10C  taken using a scanning electron microscope (SEM); 
           [0049]      FIG. 11  schematically shows the first embodiment of a Raman spectrometer according to the present invention in one mode of operation; 
           [0050]      FIG. 12  schematically illustrates the process of surface enhanced Raman spectroscopy used for various embodiments of the present invention; 
           [0051]      FIG. 13A  schematically shows plasmon field strength on a metallic sphere; 
           [0052]      FIGS. 13B to 13G  schematically show plasmon field strengths in a perfect spherical void for plasmons of varying angular momentum; 
           [0053]      FIG. 14A  shows a reflection spectrum for different thickness truncated spherical voids in a gold metallic film used in various embodiments of the present invention; 
           [0054]      FIG. 14B  shows plasmon modes for different thickness truncated spherical voids in a gold metallic film used in various embodiments of the present invention; 
           [0055]      FIG. 15  shows data indicating how the reflectivity of a gold metallic film of varying thickness varies according to the wavelength of incident optical radiation and for different angles of incidence, polarisation and metallic film orientation; 
           [0056]      FIG. 16A  is a schematic illustration of a plasmon formed in a void by coupling of optical radiation from a waveguide formed in a substrate for use in various embodiments of the present invention; 
           [0057]      FIG. 16B  is a schematic illustration of a plasmon formed in a void decaying to generate optical radiation in the waveguide shown in  FIG. 16A ; 
           [0058]      FIG. 17A  is a schematic illustration of a combined void and metal sphere for enhancing Raman signal in various embodiments of the present invention; 
           [0059]      FIG. 17B  is a schematic illustration of a microcavity formed by a void and a reflector for selectively enhancing Raman signal in various embodiments of the present invention; 
           [0060]      FIG. 17C  is a schematic illustration of a void bounded by an overhanging layer for enhancing Raman signal in various embodiments of the present invention; 
           [0061]      FIG. 17D  is a schematic illustration of a void bounded by an over-etched layer for enhancing Raman signal in various embodiments of the present invention; 
           [0062]      FIG. 18  is an optical device for filtering optical radiation incorporating a substrate according to an embodiment of the present invention; and 
           [0063]      FIG. 19  is a flow diagram illustrating a method of using the optical device of  FIG. 18 . 
       
    
    
     DETAILED DESCRIPTION 
       [0064]      FIG. 1  shows a conventional Raman spectrometer  100 . For example, the spectrometer  100  may comprise various elements of the in Via range of Raman microscopes available from Renishaw plc of Wotton-under-Edge, Gloucestershire, UK. The spectrometer  100  comprises an optical source  120 , a spectral analyser  180  and input channel optics  160  for collecting Raman scattered radiation  142  and directing it to the spectral analyser  180 . The optical source  120  generates a beam of optical radiation  122  which is filtered by a first filter  124 . The filtered optical radiation  122  is directed by beam splitter  126  on to a sample  140 . Raman scattered radiation  142  generated by the sample  140  is collected by input channel optics  160  for analysis by the spectral analyser  180 . 
         [0065]    The input channel optics  160  comprises a microscope objective lens  162  a second filter  164  and a lens  166 . The microscope objective lens  162  has a high numerical aperture (typically 0.4 or more) in order to gather as much Raman scattered radiation  142  as possible from the sample  140 . The second filter  164  is designed to block any reflected optical radiation  122  that is not Raman scattered. The lens  166  focuses the Raman scattered radiation  142  in to the spectral analyser  180 . 
         [0066]    The spectral analyser  180  comprises a spectrum separator  182  and a CCD detector  184 . The spectrum separator  182  spatially separates different frequencies of Raman scattered radiation  142 . A rotating grating (not shown) may be used to sweep different wavelengths of Raman scattered radiation  142  across an aperture placed in front of the CCD detector  184 . The CCD detector  184  is cooled in order to be able to detect low levels of Raman scattered radiation  142 . Other parallel or single channel detectors may be used. 
         [0067]    The microscope objective lens  162  needs to be placed close to the sample  140  in order to collect as much Raman scattered radiation  142  as possible. The microscope objective lens  162  collects Raman scattered radiation  142  from an area having a diameter of about Δ 1 . Typically, Δ 1  is less than 10 micrometers. Additionally, the microscope objective lens  162  needs to be placed close to the sample  140 . The microscope objective lens  162  and the sample  140  are separated by a distance L 1  which is typically less than 1 mm. 
         [0068]      FIG. 2  shows a Raman spectrometer  200  according to a first embodiment of the invention. The spectrometer  200  comprises a source/detector package  290  and a substrate  240 . The source/detector package  290  comprises an optical source  220  and a first filter  224  for filtering optical radiation  222  generated by the optical source  220 . The package  290  also includes input channel optics  260  and a spectral analyser  286 . 
         [0069]    The input channel optics  260  comprises a first lens  262  for gathering Raman scattered radiation  242  and a second filter  264  for rejecting any non-Raman scattered radiation. The input channel optics directs Raman scattered radiation to the spectral analyser  286 . 
         [0070]    The source/detector package  290  is configured to direct optical radiation  222  on to the surface of the substrate  240  and to collect Raman scattered radiation  242  that is generated by a sample that is placed proximal to the surface of the substrate  240 . The substrate  240  comprises a support layer  244  with a metallic film  246  formed thereon. The metallic film  246  comprises a plurality of voids  248 . The voids  248  generate and confine surface plasmons that couple energy from the optical radiation  222  to a sample material (not shown). The plasmons also convert scattered energy emitted from the sample material into Raman scattered radiation  242 . The plasmons give rise to a surface enhanced effect which increases significantly the amount of Raman scattered radiation  242 . This in turn means that the optical radiation  222  does not necessarily need to be tightly focused in order to generate a significant Raman signal. Additionally, it also allows use of a lens  262  which need not have a high numerical aperture. 
         [0071]    The focal spot size of the optical radiation  222 , Δ 2 , can be greater than 100 micrometers. This further enhances the Raman scattered radiation  242  since it enables a large number of sample material molecules to be illuminated at any one time. Moreover, as will be seen later on, careful design of the size and shape of the voids  248  enables the direction at which the Raman scattered radiation  242  emerges to be controlled and predicted so that appropriately positioned small solid angle collection optics is capable of collecting a high proportion of the Raman scattered signal. 
         [0072]    The optical source  220  can be a small laser diode having an output power of several tens of milliwatts. A laser diode array may also be used. The input channel lens  262  is separated from the substrate  240  by a distance L 2 . Since the lens  262  need not have a high numerical aperture it can be separated from the substrate  240  by distances of 1 cm or more. Preferably, the lens  262  (or an alternative optical radiation gathering aperture such as, for example, a fibre optic) will have a numerical aperture of less than 0.4. More preferably, the numerical aperture will be less than 0.1. This allows the Raman spectrometer  200  to be used to analyse fluids (liquids/gasses) flowing over the substrate  240 . 
         [0073]    The spectral analyser  286  comprises apparatus for spectral separation of the Raman scattered radiation  242  and a detector for measuring the Raman scattered radiation  242 . In this embodiment, the spectral analyser  286  comprises a fixed grating and an array of diodes (not shown) for detecting the spectral components of the Raman scattered radiation  242 . It is understood that conventional scanning spectrum separators may be used to detect Raman scattered radiation  242 . For example, a precision grating stage and optionally a detector from Renishaw plc&#39;s in Via Raman microscope range may be used. However, an advantage of the present embodiment is that the spectrometer  200  can be made to be ultra compact and portable. In addition reliability and detection speed are improved with respect to conventional spectrometers since it is not necessary to use a mechanically operated spectrum separator to sweep across the range of Raman scattered radiation wavelengths. 
         [0074]      FIG. 3  shows a Raman spectrometer  300  according to a second embodiment of the invention. The spectrometer  300  comprises an optical source  320 , a substrate  340  and a detector package  380 . 
         [0075]    The optical source  320  comprises a laser diode. The laser diode generates a beam of optical radiation  322  that is filtered by a first filter  324  to provide a monochromatic beam. The optical radiation  322  is coupled in to an optically transparent support layer  344  of the substrate  340 . A blazed grating is written in to the support layer  344  for coupling the optical radiation  322  from the support layer  344  in to a metallic film  346  formed on the support layer  344 . Optical radiation  322  excites plasmons in voids  348  that are formed in metallic film  346 . 
         [0076]    Sample material is placed in the voids  348  and excites Raman scattered radiation  342  in response to the plasmons generated by the optical radiation  322 . The Raman scattered radiation  342  is emitted from the metallic film  346  in a direction that depends upon the shape and size of the voids  348 . Raman scattered radiation  342  is captured by the detector package  380  and converted in to a Raman signal that represents the spectrum of the Raman scattered radiation  342 . Raman scattered radiation  342  is captured by a lens  362  which is separated from the substrate  340  by a distance L 3 . L 3  can be a distance greater than 1 cm. Raman scattered radiation collected by the lens  362  is filtered by a second filter  364  used to reject non-scattered light emerging from the substrate  340 . The filtered Raman scattered radiation is converted by a spectral analyser  386  in to a Raman signal. 
         [0077]    A spectral analyser  386  comprises a spectrum separator. In this case, the spectrum separator includes a fixed grating which separates the Raman scattered radiation  342  into various spectral components. The spectral components are angularly separated and impinge upon a diode array contained within the spectral analyser  386 . Each diode of the diode array is used to measure a spectral component of the Raman scattered radiation  342 . 
         [0078]    Electronic circuitry coupled to the diode array logs the spectrum for the Raman scattered radiation  342 . The electronic circuitry (not shown) can be coupled to a computer system for logging and manipulating the Raman spectrum data. Software may be provided to identify a particular type of substrate material in dependence upon the measured Raman spectrum. 
         [0079]      FIG. 4  shows a Raman spectrometer  400  according to a third embodiment of the invention. The Raman spectrometer  400  comprises an optical source  420  for generating optical radiation  422 . The optical radiation  422  is filtered by a first filter  424  and guided in to an optically transparent support layer  444  formed in a substrate  440 . The optical radiation  422  couples in to a metallic film  446  formed upon the support layer  444  over a distance of Δ 4 . The distance Δ 4  can be greater than 100 micrometers. 
         [0080]    Optical radiation  422  excites plasmons in voids  448  that are formed in the metallic film  446 . The plasmons couple energy to sample materials that are located near the voids  448 . The excited sample material gives rise to Raman scattered energy that couples via plasmons back in to the optically transparent support layer  444 . The support layer  444  acts as a waveguide that guides Raman scattered radiation  442  through the support layer  444 . 
         [0081]    Detector package  480  is provided to detect the Raman scattered radiation  442  that emerges from the support layer  444 . Detector package  480  comprises input channel optics  460  and a spectral analyser  486 . The input channel optics  460  comprises a lens  462  and a second filter  464  that is used to reject elastically scattered photons generated by the optical source  420 . The spectral analyser  486  comprises a fixed grating and a diode array. Each of the diodes in the diode array is used to detect a spectral component of the Raman scattered radiation  442 . 
         [0082]    Electronic circuitry (not shown) gathers data from each of the diodes in the diode array in order to reconstruct a Raman spectrum. The electronic circuitry can be configured to provide data relating to the Raman spectrum to a computer system for further analysis, identification or storage. For example, software running on such a computer system may be used to identify a particular sample material according to the measured Raman spectrum. 
         [0083]      FIG. 5  is a flow diagram illustrating method  500  of obtaining a Raman spectrum from a sample material. The method  500  can be used in conjunction with the Raman spectrometers described in connection with  FIGS. 2 to 4 . 
         [0084]    Step  502  comprises flowing a fluid containing a sample material across the surface of a substrate that contains a plurality of voids. 
         [0085]    Step  504  is a step of activating an optical source to generate optical radiation for generating surface plasmons that are confined by the voids. The surface plasmons excite an enhanced Raman scattered radiation signal from the sample material. 
         [0086]    Step  506  is a step of operating a spectral analyser to determine a Raman spectrum of the Raman scattered radiation generated in response to the activation of the optical source by the sample material. Operation of the spectral analyser may entail rotating a grating and recording a signal from a single photodetector. Alternatively, a photo diode array may be used with a fixed spectral separator. 
         [0087]    Step  508  is a decision step. The decision step entails deciding whether further Raman spectra are required. This operation may for example be pre-programmed into a computer system which is operable to generate a plurality of Raman spectra and to control a Raman spectrometer. Where such a computer system determines that further spectra are to be obtained then the method moves on to step  510 . Otherwise the method is ended. 
         [0088]    Step  510  is a step at which the electric potential of the metallic film is varied. By varying the electric potential applied to the metallic film the physical properties of the sample material can be changed. Chemical reactions of an adsorbed species can be initiated at the substrate surface at a specific bias applied potential. Subsequently, variations in the adsorbed molecules can be tracked from a time sequence of their Raman spectra, obtained in real time using fast detection. 
         [0089]    Once the potential of the metallic film has been incrementally changed, the method moves again to step  506  so that a further Raman spectrum can be obtained for the sample material which will be subject to a modified electric potential. 
         [0090]      FIG. 6  shows Raman spectra  600  of a sample material containing benzenethiol obtained using the Raman spectrometer of  FIG. 2 . The Figure shows a set of Raman spectra obtained from benzenethiol placed on a substrate having a gold metallic film incorporating a plurality of voids. The voids had a truncated spherical shape 600 nm in diameter. Various thicknesses of films were used to produce the curves A to H shown in this Figure: A—100 nm; B—160 nm; C—220 nm; D—280 nm; E—340 nm; F—460 nm; G—52 nm; and H—400 nm. The spectra indicate how by varying the properties of the voids large enhancements of the Raman cross section can be provided. For a flat gold surface no signal was observed at all. However, as the physical properties of the voids were changed a maximum intensity enhancement of some 10 4  was observed. Moreover, when the substrate was placed in a standard Raman spectrometer to obtain the results, the integration time for deriving each spectrum was only 50 milliseconds as compared to a standard conventional integration time of 5 seconds. 
         [0091]      FIG. 7  shows a set of Raman spectra of pyridine obtained with different electric potentials applied to the metallic film in solution. Raman spectra curves A-G are shown vertically offset with respect to each other for clarity. The Raman spectra  620  were obtained using the Raman spectrometer shown in  FIG. 2  operated according to the method shown in  FIG. 5 . The Raman spectra are enhanced by the effect of the structured substrate. In this case, by a factor of some 10 5 . As the electric potential applied to the metallic film is varied, it is noticeable that the spectra evolved to develop clearly defined sharp enhanced peaks. The main peaks in the curve when a potential of −1.0 volt is applied to the metallic film derive from the large number of molecules in solution (curve G). New peaks are observed to appear at critical potentials from 0.2 volts to −0.2 volts (curves A-C) which derive from just a few molecules adsorbed on the substrate, and which show the initiation of their chemical reaction directly observed as a change in molecular structure. 
         [0092]      FIGS. 8A and 8B  show modelled data indicating predicted Raman signal enhancement factors for substrates incorporating metallic films having a plurality of voids. The predicted enhancement factors are calculated using the following equation [4]: 
         [0000]      ∈ i   H   l ( k   m   a )[ k   i   aJ   l ( k   i   a )]′=∈ m   J   l ( k   i   a )[ k   m   aH   l ( k   m   a]′   (1) 
         [0000]    where J l  and H l  are spherical Bessel and Hankel functions, and the prime denotes differentiation with respect to the argument (ka). ∈ i  and ∈ m  are the dielectric constants inside and outside the sphere, with k i =√{square root over (∈ i )}ω/c and k m =√{square root over (∈ m )}ω/c the corresponding wave numbers. We take ∈ i =1 and assuming that the external material is an “ideal” metal with ∈ m (ω)=1−ω p   2 /ω 2 , where ω p  is the three dimensional plasmon frequency. Where frequencies are expressed in units of ω p , the solutions to Equation (1) for a sphere then depend only on the angular momentum quantum number 1, and the normalised sphere radius R=aω p /c. Symmetry requires that they are degenerate with respect to the azimuthal quantum number, m. 
         [0093]    Known tabulated complex dielectric constants for various metals were taken from the established literature. Equation (1) is the denominator for the rate of plasmon interactions. An estimate of the enhancement is produced by taking the inverse of the mismatch of this equation at each wavelength. This is an estimate because if Equation (1) is satisfied exactly for both real and imaginary parts, an infinite enhancement is predicted. In practice, the imaginary part of Equation (1) is never exactly satisfied, thus limiting the maximum enhancement. Use of such theoretically-derived estimates is relatively well respected by the scientific community. 
         [0094]      FIG. 8A  shows predicted enhancement factors for a variety of different metals in which the angle and momentum of the plasmons is confined to the l=1 mode, where 1 is the angular momentum quantum number. 
         [0095]      FIG. 8   b  shows predicted Raman enhancement factors for various metals in which voids confine plasmons to the l=2 mode. 
         [0096]    Both  FIGS. 8A and 8B  indicate that by carefully selecting the size of the voids to match the plasmon modes that form in the voids, enhanced coupling can be obtained beyond that already found from our experiments. Enhancement factors ranging from about 10 9  to about 10 15  are predicted from the theoretical studies. 
         [0097]      FIG. 9  is a flow diagram  700  illustrating a method of making a substrate having an enhanced efficiency of coupling optical energy to surface plasmons at a predetermined wavelength of optical radiation. The method relies upon using experimental and theoretical studies in order to optimise the performance of the substrate for any particular application by ensuring that voids provided in a metallic film give rise to strong plasmon generation for a particular desired wavelength of incident optical radiation. 
         [0098]    Step  702  entails selecting a wavelength and a metal type for a particular application. Where the substrate is to be used for Raman spectroscopy this will depend on the sample material that is to be used. For example, the sample material will often have known peaks in its Raman spectra that are generally stronger than others. In this case, the wavelength can thus be selected in order to excite the various Raman spectral features of most interest. Further, the metal type may be selected in order to provide for minimal reactivity with the sample material so as to ensure that the spectral properties derive solely from the sample material and not from a combination of the sample material and the metal used to form the substrate. 
         [0099]    Step  704  requires the matching of the wavelength selected for the sample material to the available void sizes that can be fabricated. In the technique used to manufacture the substrates according to this invention, a predetermined range of materials for forming the voids may be available. For example, the latex spheres used to manufacture the voids may only be available with a predetermined number and range of sizes. In order to form a matrix, one size that best matches the properties of the voids to the size of a void that can be made needs to be selected. For example, 700 nanometer diameter latex spheres are readily available and these can be used to form the voids. 
         [0100]    Step  706  involves ascertaining the thickness of the film needed to produce the desired optical response. Ascertaining the optimised thickness involves using the data shown in  FIG. 14B  in a normalised form to determine at what wavelength the localised plasmon resonance occurs for a particular void diameter. Since it is desirable to tune the exciting wavelength (and/or the SERS emission wavelength) to the localised plasmons, the film thickness may be selected using this technique. 
         [0101]    Step  708  involves calculating the charge that needs to be used to provide the metallic film having the optical characteristics desired for use in the particular application for which the substrate is designed. The applicants have calibrated the charge/unit area required to grow films of particular thickness with particular void size. However, this calculation can also be derived from first principles by associating the deposition of each metal with a certain number of electrons, and then calculating from the geometry of the voids how many metal atoms need to be deposited to occupy a certain thickness. 
         [0102]    Step  710  involves depositing latex spheres upon a substrate base to form a template. The technique of depositing the latex spheres and subsequently forming the metallic film on the substrate is described by the applicant in References 7, 10 and 11. The content of References 7, 10 and 11 are hereby incorporated herein by reference in their entirety. 
         [0103]    Step  712  involves introducing an electrolyte solution to surround the latex spheres that form the template. The electrolyte solution comprises ions of the metal type previously chosen to form a metallic film. The electrolyte solution permeates the template. 
         [0104]    At step  714  the electrolyte solution is electrolysed. A predetermined charge corresponding to that previously calculated is passed through the solution so that the metallic ions come out of solution and form the metallic film. The amount of charge determines the thickness of the metallic film that is deposited. 
         [0105]    Step  716  the latex sphere template is dissolved using an organic solvent. Dissolving of the latex sphere template leaves a metallic substrate including voids formed where the latex spheres previously existed. 
         [0106]    At step  718  the substrate is rinsed and dried in order to remove any traces of organic solvent and to provide a clean optically active surface for the metallic film. 
         [0107]    Optionally, following the manufacture of various substrates, they can be coated with various sample materials. This allows ready made substrates to be provided that can be used to analyse specific sample materials. Various organic materials may be provided with substrates that selectively bind to specific target molecules. For example, various oligonucleotides (fragments of DNA or RNA) which target specific DNA or RNA sequences for selective binding may be provided along with the substrates. 
         [0108]      FIG. 10A  shows a schematic illustration of a plasmon energy states  752 ,  754  formed in a void  748 . The void  748  is defined by a void surface  750  that is formed in a metallic film  746 . The voids  748  are shaped like part-spherical dishes of metal, and may be formed by electrochemically growing metal around a latex spherical former. The plasmons, which are electromagnetic modes, sit predominantly localised inside the spherical voids. Once the plasmons are excited, they decay either by radiating light or by transferring their energy to individual electrons in the surface  750  of the metal. 
         [0109]    Void surfaces  750  can be designed so as to obtain plasmon resonances at a particular angle of incidence, based on the physical parameters of a metallic film. Light of a particular wavelength couples to localised plasmons in the voids only at particular angles of incidence, which can be predicted. The coupling depends upon the thickness of the film, the diameter of the spherical void, the type of metal and the optical polarisation. 
         [0110]      FIG. 10B  shows a schematic illustration of a void  748  having a truncated spherical shape. 
         [0111]      FIG. 10C  shows a perspective view of a metallic film  746  including a plurality of voids  748 . The metallic film  746  can be incorporated in a substrate used in various embodiments of the invention. 
         [0112]      FIG. 10D  shows a plan view of the metallic film  746  shown in  FIG. 10C . The plan view was obtained by imaging the metallic film  746  using a scanning electron microscope. The diameter of the latex spheres that were used as a template to form the metallic film  746  was 700 nanometers. 
         [0113]      FIG. 11  schematically shows the Raman spectrometer  200  shown in  FIG. 2  in one mode of operation. Optical radiation  722  is focussed through a first lens  762  onto a metallic film  746 . The metallic film  746  comprises a plurality of voids  748 . A fluid containing sample material flows over the metallic film  746  in the direction of the arrow  756 . Raman scattered radiation  742  is generated by the sample material. The Raman scattered radiation  742  is collected by a second lens  766  and subsequently analysed to derive the Raman spectrum. The emission of the surface enhanced Raman scattered light is at a different angle (θ 2 ) from the incident optical radiation (θ 1 ). The metallic film  746  can be engineered to provide a spectrometer in which it is not necessary to use high numerical aperture lenses. High numerical aperture lenses have a short working distance from the sample so as to capture light emerging from the sample from as many angles of emission as possible. Embodiments of the invention enable larger areas of the substrate to be examined simultaneously. This also increases the Raman signal that is observed because more photons are gathered. Furthermore, the Raman signal can be collected by optics that do not necessarily need to be placed close to the substrate surface. 
         [0114]    We have also shown that it is possible directly to observe real time changes in the chemistry of a sample material monolayer at the surface of the substrate. The substrate is placed in a solution containing sample material. The optical radiation passes through the solution and excites the sample material proximal to the substrate. By applying a potential to the solution by placing an electric potential on the substrate surface, molecules of sample material can be selectively electrochemically bound to the surface. Previously this was impractical because it was difficult to separate Raman scattered photons generated close to the surface from Raman scattered photons generated by molecules in solution remote from the surface. However, now since the surface molecules provide an enhanced Raman signal, Raman signal arising from sample material near the surface dominates, swamping any Raman signal arising from the body of the solution away from the surface. 
         [0115]    The invention therefore enables real time tracking of the progress of surface chemical reactions with the possibility of initiating the surface chemical reactions using laser pulses to excite sample material molecules via plasmons generated in the voids. The study of small numbers of molecules contained in a single void is also made possible by the enhanced Raman signal. In addition, our theoretical studies predicted that enhanced Raman signals are also derivable using a platinum-based substrate or a palladium-based substrate. This allows for the direct study of catalysis. 
         [0116]      FIG. 12  schematically illustrates the process of surface enhanced Raman spectroscopy. A photon of optical radiation  822  is incident on the metallic surface of the substrate  840 . The photon is incident on the metallic surface and gives rise to an electromagnetic disturbance in the form of a surface plasmon  852 . The surface plasmon  852  couples energy from the surface of the substrate  840  into sample material  858 . The plasmon energy couples with the energy of a phonon and converts into a further surface plasmon  854 . The plasmon  854  subsequently transfers energy to a Raman scattered photon  842 . 
         [0117]    A flat metal film does not efficiently convert incident light to plasmons or plasmons into emitted photons. The voids of the present invention, however, provide a controlled way of doing this by careful choice of the void size and shape. 
         [0118]      FIG. 13A  schematically shows the plasmon field strength on a metallic sphere. The plasmon intensity on the surface of the sphere, and in its vicinity, is not high and decays only slowly. This means that plasmons generated on the surface of metallic spheres are not best suited to coupling energy from incident photons to any sample material that is placed near to the spheres in order to obtain an enhanced Raman signal. This is one reason why various roughened surfaces used in existing SERS devices are less effective. 
         [0119]      FIGS. 13B to 13G  schematically show plasmon field strengths for a perfect spherical void.  FIGS. 13C to 13G  show how the plasmon field strengths appear as different modes depending on the angular momentum (l,m) of the plasmons that are excited. In each case it can be seen that at least one high field strength “hot spot” develops, as indicated by the light coloration regions shown within the voids. The high field strength enables energy to be coupled efficiently from incident optical radiation into sample materials that are placed in or near to the voids. 
         [0120]      FIG. 14A  shows a reflection spectrum for different thickness voids as the thickness increases from near zero (thin) to about 700 nanometres (thick). Optical radiation is incident normal to the substrate surface. 
         [0121]      FIG. 14B  shows plasmon modes for different thickness truncated spherical voids in a gold metallic film. The metallic film is the same as that used to provide the results shown in  FIG. 14A . The plasmon modes have been extracted from the reflectivity data and their energies are compared with the energy of the plasmon on a flat gold film, i.e. a two-dimensional (2D) plasmon. The energies of the plasmons are also compared to those of a perfect spherical void, i.e. a zero-dimensional (0D) plasmon, for different angular momentum values l=1 and l=2. The localised plasmons (known as Mie modes, M 1  and M 2 ) start out with an energy equal to the 2D plasmons for a very shallow void. As the void gets thicker the energy drops, tending to the energy of a complete spherical void as the thickness approaches 700 nanometers. This is clearly seen in the data, which also shows the theoretical limits (2D and 0D), and the experimental data moving smoothly between them. This information is useful as it enables tailored metallic films to be produced that efficiently couple optical radiation of a particular wavelength into plasmons. 
         [0122]    Two additional modes are also seen in the data. These are known as a localised mode (L 3 ) and a Bragg mode (B 4 ). The localised mode (L 3 ) arises from 2D plasmons which move along the flat gold surface in between the voids. These can become localised in the gaps above the voids rather than on the gold in between the voids. It is expected that this mode will also give rise to an enhanced Raman signal. 
         [0123]      FIG. 15  shows data indicating how the reflectivity of a gold metallic film of varying thickness varies according to the wavelength of incident optical radiation and for different angles of incident polarisation and metallic film orientation. 
         [0124]    The substrate comprises metallic film made of gold. The metallic film was some 5 mm long. Voids formed in the metallic film varied in depth from zero (i.e. flat) at the zero mm position to 700 nanometers at the 5 mm position. The angle φ represents the angle of rotation of the sample about the surface normal. The surface has sixfold symmetry due to the hexagonal packing of the truncated spherical voids, and hence rotation was made between 0° and 30°. The angle θ corresponds to the angle of incidence of the optical radiation with respect to the surface of the substrate. Normal incidence is at 0° and measurements were made up to 27° incrementally in steps of 3°. Measurements were made for both the transverse electric and the transverse magnetic field. Further details of the optical set-up for obtaining these results can be found in Reference 8. 
         [0125]    The data indicates that whereas a perfectly spherical void has no angular dependence, truncated spherical voids give rise to localised plasmons that emit at different wavelengths in different directions. Each mode changes wavelength with angle in a way that can be predicted from a comparison with experimental results or from modified experimental results derived from theory. By truncating spherical voids a coupling together of dipole, quadruple, hexapole, etc. plasmons occurs which shifts the coupled plasmon modes to a higher energy and introduces angular dependence. The presence of a strong optical field for some of these modes on a metal boundary (for example (l, m)=(1, 0), (2, 0)) is what allows light impinging on the structure to couple strongly to the localised plasmons. This process can be modelled. (For example, see  FIGS. 8A and 8B .) Moreover, using the applicant&#39;s data, it has been possible to produce substrates with voids that confine plasmons in both platinum and nickel. Both of these materials are interesting because of their catalytic properties. 
         [0126]      FIG. 16A  shows a schematic illustration of a plasmon formed in a void by coupling of optical radiation from a waveguide formed in a substrate. The substrate  940  comprises a support layer  944  made using low refractive index glass. A high refractive index glass waveguide layer  947  is formed over the support layer  944 . Metallic film  946  incorporating a plurality of voids  948  is formed on the waveguide layer  947 . Optical radiation  922  is guided in the waveguide layer  947 . 
         [0127]    Where the voids  948  are in close proximity to the waveguide layer  947  optical radiation  922  can couple to the surface of the voids  948 . This coupling generates plasmons in a void  948 . The plasmons  952  are able to couple to sample material in the voids  948  and generate Raman scattered radiation  942 . Some of the Raman scattered radiation  942  couples back into the waveguide layer  947  and can be detected remote from the voids  948 . 
         [0128]    By combining the voids with an optical waveguide, either the input optical radiation or the output surface enhanced Raman signal, or both, can be injected/collected through the waveguide. In a first version, optical radiation is fed in through the optical waveguide and couples to the localised plasmons via evanescent coupling. The applicants have made such a device using a gold metallic film formed on an indium tin oxide (ITO) layer forming a waveguide over a glass support layer. 
         [0129]      FIGS. 17A to 17D  show various schemes for improving the coupling of optical radiation into sample materials by modifying the geometry of the voids. In  FIG. 17A  a metal sphere  1049  is placed in the void  1048 . The metal sphere can be a gold, silver or copper sphere which is either solid or which has a dielectric core. Theoretical predictions indicate that use of such a sphere  1049  will give rise to a further enhanced Raman signal. 
         [0130]      FIG. 17B  shows a mirror device  1149  placed above the void  1148  in order to form a microcavity. The microcavity enhances the Raman signal by selecting certain wavelength bands for amplification. By adjusting the length of the cavity, a particular set of wavelength bands can be amplified. The mirror device  1149  can be a dielectric Bragg reflector, or a thin metallic layer. Additionally, this geometry allows MEMS devices to be constructed in conjunction with the substrate. 
         [0131]      FIGS. 17C and 17D  illustrate how electrochemically grown metal over-layers can be provided to produce a modified void. In  FIG. 17C  gold layer  1246  is provided with an overhanging silver layer  1249 . In  FIG. 17D  gold layer  1346  is provided with an over-etched silver layer  1349 . 
         [0132]      FIG. 18  shows an optical device  1400  for filtering optical radiation  1422 . The optical device  1400  comprises a substrate  1440  having a metallic film  1446  that includes a plurality of voids  1448 . The voids  1448  are designed to emit radiation of a particular wavelength at a particular angle. The optical device  1400  incorporates an optical aperture  1470  for blocking radiation which does not emerge from the substrate  1440  at a particular predetermined angle. Only radiation  1442  having a predetermined wavelength is able to emerge from the optical device  1400 . Thus, the optical device  1400  acts to filter the optical radiation  1422 . 
         [0133]      FIG. 19  is a flow diagram  1500  illustrating a method of using the optical device  1400  shown in  FIG. 18 . 
         [0134]    Step  1502  requires the generation of radiation which is to be filtered. The optical radiation is provided to the optical device. 
         [0135]    Step  1504  entails reflecting of the radiation to be filtered from a substrate. The substrate disperses the radiation according to its wavelength. Radiation of a particular predetermined wavelength leaves a surface of the substrate at a particular predetermined angle. 
         [0136]    Step  1506  comprises collimating reflected radiation in order to remove the components of the incident radiation that do not emerge from the substrate at a particular predetermined angle. In one example, a pinhole or the like may be used to selectively block dispersed optical radiation. The angular dispersion and collimation of the radiation reflected from the substrate therefore enables the incident optical radiation to be filtered. 
         [0137]    Whilst the invention has been described in relation to various embodiments, many variations will be envisaged by the skilled person. For example, one possibility is to take an existing fibre optic probe and create a semitransparent substrate on top of this so that light can couple from the fibre optic onto the substrate and so that SERS photons can be detected in a direction back down the fibre optic. Such a probe can be fabricated as an immersible probe without a microscope objective or other lens. Moreover, those skilled in the art will realise that various features of different embodiments may be combined as necessary to obtain still further embodiments of the invention. 
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