WO2005047832A1 - Method of spectroscopy - Google Patents

Method of spectroscopy Download PDF

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Publication number
WO2005047832A1
WO2005047832A1 PCT/GB2004/004693 GB2004004693W WO2005047832A1 WO 2005047832 A1 WO2005047832 A1 WO 2005047832A1 GB 2004004693 W GB2004004693 W GB 2004004693W WO 2005047832 A1 WO2005047832 A1 WO 2005047832A1
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Prior art keywords
sample
excitation source
spectroscopy
vibrational mode
excitation
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PCT/GB2004/004693
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French (fr)
Inventor
David Klug
Jason Donald Palmer
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Imperial College Innovations Limited
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Priority to EP04798417A priority Critical patent/EP1685370A1/en
Publication of WO2005047832A1 publication Critical patent/WO2005047832A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/10—Arrangements of light sources specially adapted for spectrometry or colorimetry
    • G01J3/108—Arrangements of light sources specially adapted for spectrometry or colorimetry for measurement in the infrared range
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]
    • G01N2021/655—Stimulated Raman
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention relates to a method of spectroscopy, in particular multidimensional spectroscopy.
  • information obtained from the second excitation pulse differs from the information obtained from the first excitation pulse providing an extra dimension.
  • a Fourier transformation is applied to the time spectrum from each excitation pulse to obtain a respective frequency spectrum.
  • the frequency spectra are plotted on orthogonal axes to form a surface. Peaks on the surface provide additional information concerning interactions within the sample.
  • 2D-NMR plots can be used to determine molecular structure and provide unique, characteristic features ("fingerprints") for identifying components in a solution.
  • fingerprints unique, characteristic features
  • 2D-NMR suffers from a lack of sensitivity, with detection limits typically on the order 10 15 -10 18 molecules.
  • 2D-NMR provides only limited resolution in the time domain.
  • techniques analogous to those used in 2D-NMR spectroscopy have been adopted in 2D vibration or infrared (IR) spectroscopy, where vibrational modes of an atom or molecule are excited.
  • Fig. 1 shows an apparatus for performing a method of spectroscopy according to the present invention.
  • the invention relates to a method of spectroscopy relying on excitation of a vibrational mode of atoms or molecules in a system for example by excitation by an infrared excitation source. Interactions between vibrations in the system allow two or more dimensional information to be obtained with suitable excitation regimes.
  • the present invention relies on heterodyne detection allowing the output signal to vary linearly with the concentration of the sample. As a result much lower concentrations can be analysed than with, for example homodyne detection where there is a quadratic dependence on concentration and hence a vanishingly small signal for low concentrations.
  • the invention relies on a heterodyning field either from an external source or relying on a field generated by local oscillators within the sample such that linear terms dominate quadratic terms in the output signal.
  • Fig. 1 the apparatus is shown generally as including a sample 10, excitation sources comprising lasers emitting radiation typically in the infrared band and a detector 14.
  • Tunable lasers 12 and 18 emit excitation beams of respective wavelengths/wavenumbers 3164cm “1 and 2253cm "1 which excite one or more vibrational modes of the molecular structure of the sample and allow multi-dimensional data by tuning the frequencies or providing variable time delays.
  • a third, fixed frequency beam at 795nm is generated by a third laser 16 to provide an output or read out in the form of an effectively scattered input beam, frequency shifted (and strictly generated as a fourth beam) by interaction with the structure of sample 10.
  • the detected signal is typically in the visible or near infrared part of the electromagnetic spectrum eg at 740nm, comprising photons of energy not less than leV. .
  • the sample is excited by successive beams spaced in the time domain.
  • any appropriate multi-dimensional spectroscopic technique can be adopted, for example by varying the input in the frequency domain rather than the time domain.
  • any number of dimensions can be obtained by additional pulses in the time domain or additional frequencies in the frequency domain.
  • a transmission scheme is shown, a reflection scheme (where the sample reflects the detected beam) can be adopted where appropriate, for example in the case of surface deposited samples.
  • parameters of the apparatus are varied so that heterodyne detection is achieved. This can be done either by providing an external heterodyne excitation source, for example comprising a further excitation laser or broadband laser source (not shown) or by tuning the excitation laser 12 or 20 appropriately.
  • an external heterodyne excitation source for example comprising a further excitation laser or broadband laser source (not shown) or by tuning the excitation laser 12 or 20 appropriately.
  • E H o is the homodyne signal from the sample; it can be thought of as the sum electric field which is emitted by the sample component of interest.
  • E L0 is a "local oscillator" field, that is, a field of identical frequency present on the detector with a fixed phase difference ⁇ .
  • E HO 2 > which varies quadratically is with the concentration of the chemical system under study.
  • heterodyne detection a separate local oscillator is created and made to coincide in time and space on the detector. By so doing, and removing the (E L o) term byany appropriate technique which will be familiar to the skilled reader the cross term can be made to dominate the equation. With knowledge of the local oscillator strength, the output field is then linear in concentration.
  • the present invention comprises a "coherence spectroscopy”.
  • photons and molecular states comprise two halves that are separate but related. Interactions which change populations (for example, "an electron promoted to the first excited state") actually involve two interactions with the incident field, one acting on each half of the state in question. By contrast, single interactions with the incident field create what is known as a coherence, a purely quantum mechanical superposition of two molecular states.
  • the system oscillates back and forth between the two states with a frequency related to the energy difference between them. This oscillation in turn emits a field at that characteristic frequency (for instance,
  • the approach of the present invention provides a surprising level of sensitivity and, in the case of the signal field generated within the sample, relies on the fact that this is automatically a heterodyning field with the correct phase relationship.
  • heterodyning detection is implemented, as mentioned this can be obtained either by varying parameters of the sample, or of an external field generator.
  • a local oscillator field will be naturally present in the sample/solvent in the form of the non-resonant contribution inherently created.
  • the E o and cross terms can be made to dominate equation (1) and we can effectively consider the signal as heterodyned.
  • the signal is linear in concentration, and clearly far lower concentrations can be achieved before reaching the limit of detection.
  • the relative size of the local oscillator contribution can be controlled, allowing a great deal of range in the concentrations that can be examined.
  • Another way in which the relevant parameters of the sample can be controlled is by the addition of appropriate amounts of fluorescent absorptive molecules whose electronic absorption is resonant with a visible beam.
  • the absorption results in a polarisation in the molecules which radiate and serve as the local oscillator field.
  • This is a particularly useful method in that the concentration of local oscillator molecules can be precisely controlled. In that case an additional excitation source provides the required polarisation.
  • the (E L0 ) 2 term can be removed by identifying and subtracting the characteristic local oscillation signal which can be obtained in a calibration step.
  • conventional optical heterodyning can be adopted in which a local oscillator field is created external to the sample and directed to be incident on the detector with the sample field.
  • Such local oscillator generation may be by, for instance, continuum generation in a suitable crystal liquid or solid with a portion of the visible beam.
  • the specific parameters of the external signal are controlled so that the relevant terms dominate in equation (1) above, in contrast to conventional optical heterodyning systems where the linear contribution is swamped as discussed in more detail above.
  • Removal of the (E L0 ) 2 term is then simply achieved using a lock-in detector whereby a mechanical wheel with slots in it ("a chopper”) is introduced into an excitation beam.
  • the repetition rate with which the slots block the beam (reference frequency) is passed to a lock-in detector, which is basically a frequency filter - it measures the total net signal coming from the detector and extracts the component of the signal which occurs at the reference frequency. If the reference frequency is different from the repetition rate of the beam which causes the local oscillator signal, the component of the net signal due exclusively to the local oscillator is subtracted off. Then, the E L o term disappears, the E H o term is negligible, and the cross term is linear in concentration.
  • the output signal is a cone of rays containing all of the spectral information in space;
  • the detector 14 can in this case be a 2D array detector such as a charge coupled device (CCD) which captures the spectral information encoded into spatial dimensions.
  • CCD charge coupled device
  • additional dimensions are introduced either by time delays in the pulses or by frequency variations as discussed in more detail above to give yet further, fully detailed information concerning the spectrum generated by the sample.
  • Heterodyning across the frequency band is achieved by "continuum generation” whereby a spread of local oscillator frequencies is generated in the sample for example by external excitation with white light to provide a heterodyning field to interact with the broadband excitation.
  • the invention employs a geometry known as the "forward box" configuration (that is, the three beams from lasers 12, 16, 18 do not lie in the same plane) and broadband excitation beams (a large spread of input wavelengths is present in a single beam).
  • a spread of output angles is created, and a unique output direction is created for a given combination of quasi-discrete wavelengths present in the two infrared beams.
  • the spectral information is spread out into the spatial characteristics of the output beam, and the array detector effectively captures in one shot what would otherwise be built up by using narrower band excitation beams and tuning point-by-point.
  • an ultraviolet or visible excitation probes excites an electronic resonance which in turn gives rise to fluorescence caused by transitions between electronic energy levels. This is in combination with direct infrared excitation of the type discussed above. In that case the additional "read out" signal from laser 16 is not required. Tuning of input infrared and ultraviolet beams and varying time delays yields multi-dimensional data again in a manner described in more detail above, but based on a population spectroscopy.
  • the invention can be implemented in a range of applications and in particular any area in which multi-dimensional optical spectroscopy measuring, directly or indirectly, vibration/vibration coupling is appropriate, using two or more variable frequencies of light or time delays to investigate molecular identity and/or structure.
  • the techniques are particularly effective at low molecular concentration.
  • the invention improves the level of sensitivity for low-concentration detection such that gas phase and surface-deposited samples can be investigated to allow identification of components and their concentrations in complex mixtures such as proteins present in a solution, with a suitable choice of frequency range.
  • any appropriate specific component and techniques can be adopted to implement the invention.
  • at least one tuneable laser source in the infrared and at least one other tuneable laser source in the ultraviolet, visible or infrared can be adopted and any appropriate laser can be used or indeed any other appropriate excitation source.
  • a further fixed- frequency beam may also be incorporated in the case of two infrared excitation beams as discussed with reference to Fig. 1 and again any appropriate source can be adopted.
  • the sample and solvent can be of any appropriate type whereby its composition is controlled to tune the system as described in more detail above, and in any appropriate phase including gas phase and liquid/solution phase.
  • Any appropriate detector may be adopted, for example a CCD or other detector as is known from 2D IR spectroscopy techniques.
  • excitation wavelengths is generally described above as being infrared but can be any appropriate wavelength required to excite a vibrational mode of the structure to be analysed. Although the discussion above relates principally to two-dimensional analysis, any number of dimensions can be introduced by appropriate variation of the parameters of the input excitation, for example frequency, time delay/number of pulses or any other appropriate parameter.

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Abstract

A spectroscopy method and apparatus comprises an excitation source arranged to excite a vibrational mode of a sample and provide multi-dimensional spectral information by varying the excitation in a time or frequency domain. A parameter of a further excitation source or of the sample is controlled so as to provide coherence spectroscopy by ensuring that a non-resonant local oscillator field generated in the sample dominates a homodyne signal generated in the sample. As a result heterodyne detection is achieved in a manner allowing an output signal linearly dependent upon concentration providing improved sensitivity. In an alternative embodiment where heterodyning is not used, an ultraviolet or visible excitation excites an electronic resonance, whereafter fluorescence is measured.

Description

Method of Spectroscopy
The invention relates to a method of spectroscopy, in particular multidimensional spectroscopy.
A range of spectroscopic approaches are known for investigating the coupling of two or more two-level systems. One known approach is two-dimensional nuclear magnetic spectroscopy (2D-NMR). An example of such a system is described in Friebolin, "Basic one- and two-dimensional NMR spectroscopy" 2nd edition (April 1993) John Wiley & Sons. NMR relies on the interaction of magnetic nuclei with an external magnetic field, as is well known. In order to spread out crowded data in an NMR spectrum, 2D NMR has been developed. In a typical 2D-NMR scheme the sample is subjected to first and second excitation pulses separated by a delay interval. Because of interactions within the sample and in particular spin-spin coupling, information obtained from the second excitation pulse differs from the information obtained from the first excitation pulse providing an extra dimension. A Fourier transformation is applied to the time spectrum from each excitation pulse to obtain a respective frequency spectrum. The frequency spectra are plotted on orthogonal axes to form a surface. Peaks on the surface provide additional information concerning interactions within the sample.
2D-NMR plots can be used to determine molecular structure and provide unique, characteristic features ("fingerprints") for identifying components in a solution. There are a great many applications for the analysis of complex mixtures of molecules in chemistry, biology, and other disciplines. However 2D-NMR suffers from a lack of sensitivity, with detection limits typically on the order 1015-1018 molecules. In addition 2D-NMR provides only limited resolution in the time domain. In another known method of spectroscopy, techniques analogous to those used in 2D-NMR spectroscopy have been adopted in 2D vibration or infrared (IR) spectroscopy, where vibrational modes of an atom or molecule are excited. One such known technique is the so-called "pump-probe" technique as described in Woutersen et al "Structure Determination of Trialanine in Water Using Polarization Sensitive Two-Dimensional Vibrational Spectroscopy" J. Phys. Chem. B 104, 11316-11320, 2000. Further 2D-IR pump-probe experiments have been performed, for example as described in Hamm et al "The two-dimensional IR non-linear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure" Proc. Nat. Acad. Sci. 96, 2036, 1999.
According to known 2D IR systems a first, pump pulse is followed by a probe pulse and the resulting frequency spectra plotted on respective axes to provide a surface representing information about vibration- vibration interactions in the sample. Because the mathematical description of coupled two-level quantum systems is essentially identical, the analytical principles and techniques used in 2D-NMR are equally applicable in 2D IR spectroscopy. However detectivity is severely limited by input laser noise and the results show extremely small changes on a large background signal, in particular small changes in the intensity of an incident beam caused by equally small changes in the optical density of a sample. As a result there is much lower sensitivity to concentrations of the component of interest.
Another problem that arises in some instances is that the excitation and detective wavelengths are in the mid-infrared hence suffering from the problem of poorly performing detectors in that region. No existing technique provides the qualities both of high sensitivity at low concentrations or high temporal resolution down to the timescale of molecular interactions allowing a full frequency and time-result fingerprint of a given complex chemical sample. In particular multi-dimensional spectroscopies have not been implemented for the case of low concentrations.
The invention is set out in the claims.
Embodiments of the invention will now be described, by way of example, with reference to Fig. 1 which shows an apparatus for performing a method of spectroscopy according to the present invention.
In overview the invention relates to a method of spectroscopy relying on excitation of a vibrational mode of atoms or molecules in a system for example by excitation by an infrared excitation source. Interactions between vibrations in the system allow two or more dimensional information to be obtained with suitable excitation regimes. The present invention relies on heterodyne detection allowing the output signal to vary linearly with the concentration of the sample. As a result much lower concentrations can be analysed than with, for example homodyne detection where there is a quadratic dependence on concentration and hence a vanishingly small signal for low concentrations. Yet further the invention relies on a heterodyning field either from an external source or relying on a field generated by local oscillators within the sample such that linear terms dominate quadratic terms in the output signal.
Referring to Fig. 1 the apparatus is shown generally as including a sample 10, excitation sources comprising lasers emitting radiation typically in the infrared band and a detector 14. Tunable lasers 12 and 18 emit excitation beams of respective wavelengths/wavenumbers 3164cm"1 and 2253cm"1 which excite one or more vibrational modes of the molecular structure of the sample and allow multi-dimensional data by tuning the frequencies or providing variable time delays. A third, fixed frequency beam at 795nm is generated by a third laser 16 to provide an output or read out in the form of an effectively scattered input beam, frequency shifted (and strictly generated as a fourth beam) by interaction with the structure of sample 10. The detected signal is typically in the visible or near infrared part of the electromagnetic spectrum eg at 740nm, comprising photons of energy not less than leV. . In order to obtain multi-dimensional data, the sample is excited by successive beams spaced in the time domain. However any appropriate multi-dimensional spectroscopic technique can be adopted, for example by varying the input in the frequency domain rather than the time domain. Similarly any number of dimensions can be obtained by additional pulses in the time domain or additional frequencies in the frequency domain. Although a transmission scheme is shown, a reflection scheme (where the sample reflects the detected beam) can be adopted where appropriate, for example in the case of surface deposited samples.
In order to obtain the improved sensitivity of the invention, parameters of the apparatus are varied so that heterodyne detection is achieved. This can be done either by providing an external heterodyne excitation source, for example comprising a further excitation laser or broadband laser source (not shown) or by tuning the excitation laser 12 or 20 appropriately.
In either case, the manner in which the system is varied or tuned will be understood from the following discussion as will be clear to the skilled reader who can achieve the relevant objectives through routine experimentation based on this discussion. By way of definition, all the spectroscopic methods including the present invention emit a signal whose intensity can be defined as follows:
I = (EL0)2 + (EHO)2 + (ELOX EHO) cos φ (1)
Here, EHo is the homodyne signal from the sample; it can be thought of as the sum electric field which is emitted by the sample component of interest. EL0 is a "local oscillator" field, that is, a field of identical frequency present on the detector with a fixed phase difference φ. In standard homodyne detection, there is no local oscillator field, and the intensity is simply the homodyne term EHO 2 > which varies quadratically is with the concentration of the chemical system under study. In heterodyne detection, a separate local oscillator is created and made to coincide in time and space on the detector. By so doing, and removing the (ELo) term byany appropriate technique which will be familiar to the skilled reader the cross term can be made to dominate the equation. With knowledge of the local oscillator strength, the output field is then linear in concentration.
The underlying physical mechanisms exploited according to the present invention are best understood by comparing the approach adopted with typical one-dimensional spectroscopies such as absorption spectrometry, Fourier transform infrared (FTIR) spectroscopy and so forth which rely on inducing and/or measuring population state changes. In contrast to such "population spectroscopies", the present invention comprises a "coherence spectroscopy".
In the quantum mechanical picture, photons and molecular states comprise two halves that are separate but related. Interactions which change populations (for example, "an electron promoted to the first excited state") actually involve two interactions with the incident field, one acting on each half of the state in question. By contrast, single interactions with the incident field create what is known as a coherence, a purely quantum mechanical superposition of two molecular states. The system oscillates back and forth between the two states with a frequency related to the energy difference between them. This oscillation in turn emits a field at that characteristic frequency (for instance,
EHo above). It is now clear why homodyning results in a quadratic dependence detected photon. Whereas in the invention a field is introduced external to the sample (or within the sample) in order to serve as the second interaction, there is now a linear dependence on concentration: one field per sample molecule plus one from the external field results in one photon detected. We thereby define two types of spectroscopy, those manipulating coherences (coherence spectroscopies) and those manipulating populations (population spectroscopies). Coherence spectroscopies require a condition known as phase matching — a direct consequence of the conservation of energy and momentum — to identify an output direction for the beam. For a set of single, discrete input wavelengths, a unique output direction (angle) is defined.
As a result the approach of the present invention provides a surprising level of sensitivity and, in the case of the signal field generated within the sample, relies on the fact that this is automatically a heterodyning field with the correct phase relationship.
Turning to the manner in which heterodyning detection is implemented, as mentioned this can be obtained either by varying parameters of the sample, or of an external field generator.
In the case of the sample, for example a sample provided in solution, a local oscillator field will be naturally present in the sample/solvent in the form of the non-resonant contribution inherently created. By ensuring that this non- resonant contribution is significantly (say, 10 times) larger than the resonant contribution or homodyne portion of the signal, the E o and cross terms can be made to dominate equation (1) and we can effectively consider the signal as heterodyned. As such, the signal is linear in concentration, and clearly far lower concentrations can be achieved before reaching the limit of detection. Further, by varying the type of solvent, solvent concentration or volume or different thickness in the sample holder, the relative size of the local oscillator contribution can be controlled, allowing a great deal of range in the concentrations that can be examined.
Another way in which the relevant parameters of the sample can be controlled is by the addition of appropriate amounts of fluorescent absorptive molecules whose electronic absorption is resonant with a visible beam. The absorption results in a polarisation in the molecules which radiate and serve as the local oscillator field. This is a particularly useful method in that the concentration of local oscillator molecules can be precisely controlled. In that case an additional excitation source provides the required polarisation.
In intra-sample cases such as these where the local oscillator field is internally or intrinsically generated the (EL0)2 term can be removed by identifying and subtracting the characteristic local oscillation signal which can be obtained in a calibration step.
In a further approach, conventional optical heterodyning can be adopted in which a local oscillator field is created external to the sample and directed to be incident on the detector with the sample field. Such local oscillator generation may be by, for instance, continuum generation in a suitable crystal liquid or solid with a portion of the visible beam. In that case the specific parameters of the external signal are controlled so that the relevant terms dominate in equation (1) above, in contrast to conventional optical heterodyning systems where the linear contribution is swamped as discussed in more detail above. Removal of the (EL0)2 term is then simply achieved using a lock-in detector whereby a mechanical wheel with slots in it ("a chopper") is introduced into an excitation beam. The repetition rate with which the slots block the beam (reference frequency) is passed to a lock-in detector, which is basically a frequency filter - it measures the total net signal coming from the detector and extracts the component of the signal which occurs at the reference frequency. If the reference frequency is different from the repetition rate of the beam which causes the local oscillator signal, the component of the net signal due exclusively to the local oscillator is subtracted off. Then, the ELo term disappears, the EHo term is negligible, and the cross term is linear in concentration.
In a further embodiment, "multiplexing" of the type described in Muller et al, "Imaging the Thermodynamic State of Lipid Membranes with Multiplex CARS Spectroscopy" J. Phys. Chem. B. 106, 3715-3723 is achieved by the use of broadband pulses in the infrared, created by ultrafast pulses to simultaneously excite infrared transitions in the sample and the spectrals portions surrounding them. By appropriate selection of the input angles of the beams, unique directions corresponding to input frequencies can be achieved. As a result the output signal is a cone of rays containing all of the spectral information in space; the detector 14 can in this case be a 2D array detector such as a charge coupled device (CCD) which captures the spectral information encoded into spatial dimensions. Once again to obtain improved resolutions of spectra, in addition to the spatial dimensions, additional dimensions are introduced either by time delays in the pulses or by frequency variations as discussed in more detail above to give yet further, fully detailed information concerning the spectrum generated by the sample. Heterodyning across the frequency band is achieved by "continuum generation" whereby a spread of local oscillator frequencies is generated in the sample for example by external excitation with white light to provide a heterodyning field to interact with the broadband excitation.
In a particular implementation the invention employs a geometry known as the "forward box" configuration (that is, the three beams from lasers 12, 16, 18 do not lie in the same plane) and broadband excitation beams (a large spread of input wavelengths is present in a single beam). In this way, a spread of output angles is created, and a unique output direction is created for a given combination of quasi-discrete wavelengths present in the two infrared beams. Hence, the spectral information is spread out into the spatial characteristics of the output beam, and the array detector effectively captures in one shot what would otherwise be built up by using narrower band excitation beams and tuning point-by-point.
In a further, alternative embodiment where heterodyning is not used, an ultraviolet or visible excitation probes excites an electronic resonance which in turn gives rise to fluorescence caused by transitions between electronic energy levels. This is in combination with direct infrared excitation of the type discussed above. In that case the additional "read out" signal from laser 16 is not required. Tuning of input infrared and ultraviolet beams and varying time delays yields multi-dimensional data again in a manner described in more detail above, but based on a population spectroscopy.
The invention can be implemented in a range of applications and in particular any area in which multi-dimensional optical spectroscopy measuring, directly or indirectly, vibration/vibration coupling is appropriate, using two or more variable frequencies of light or time delays to investigate molecular identity and/or structure. The techniques are particularly effective at low molecular concentration. For example the invention improves the level of sensitivity for low-concentration detection such that gas phase and surface-deposited samples can be investigated to allow identification of components and their concentrations in complex mixtures such as proteins present in a solution, with a suitable choice of frequency range.
The skilled person will recognise that any appropriate specific component and techniques can be adopted to implement the invention. Typically at least one tuneable laser source in the infrared and at least one other tuneable laser source in the ultraviolet, visible or infrared can be adopted and any appropriate laser can be used or indeed any other appropriate excitation source. A further fixed- frequency beam may also be incorporated in the case of two infrared excitation beams as discussed with reference to Fig. 1 and again any appropriate source can be adopted. Similarly the sample and solvent can be of any appropriate type whereby its composition is controlled to tune the system as described in more detail above, and in any appropriate phase including gas phase and liquid/solution phase. Any appropriate detector may be adopted, for example a CCD or other detector as is known from 2D IR spectroscopy techniques.
The range of excitation wavelengths is generally described above as being infrared but can be any appropriate wavelength required to excite a vibrational mode of the structure to be analysed. Although the discussion above relates principally to two-dimensional analysis, any number of dimensions can be introduced by appropriate variation of the parameters of the input excitation, for example frequency, time delay/number of pulses or any other appropriate parameter.

Claims

Claims
1. A method of spectroscopy having as controllable parameters at least one of an excitation source parameter and a sample parameter, comprising controlling a controllable parameter to generate a local oscillator field in the sample of a magnitude to dominate a homodyne signal from the sample, exciting a vibrational mode of the sample and obtaining a spectrum of the excited sample.
2. A method as claimed in claim 1 in which the sample is excited so as to obtain a spectrum in at least two dimensions.
3. A method as claimed in claim 2 in which a sample excitation is varied in at least one of the time domain and the frequency domain to obtain the spectrum in at least two dimensions.
4. A method as claimed in any preceding claim in which the controllable parameter is a sample parameter.
5. A method as claimed in claim 4 in which the sample parameter is varied by the addition of absorptive molecules in the sample.
6. A method as claimed in any preceding claim in which the controllable parameter comprises at least one of the frequency, phase or amplitude of an external excitation source beam.
7. A method of spectroscopy comprising the steps of exciting a vibrational mode of a sample, exciting an electronic mode of a sample and measuring a change in fluorescence of the sample, in which the sample is excited so as to obtain a spectrum in at least two dimensions.
8. A method of spectroscopy comprising exciting a vibrational mode of a sample, exciting a spectral portion surrounding the vibrational mode using an excitation beam, generating a broadband local oscillator field in the sample by a broadband excitation source, and detecting a spatially resolvable output beam.
9. A spectroscopy apparatus comprising an excitation source arranged to excite a vibrational mode of a sample and a further excitation source arranged so as to generate a local oscillator field in the sample of a magnitude to dominate a homodyne signal from the sample, exciting a vibrational mode of the sample and obtaining a spectrum of the excited sample.
10. A spectroscopy apparatus comprising an excitation source arranged to excite a vibrational mode of a sample, and a sample arranged so as to generate a local oscillator field in the sample of a magnitude to dominate a homodyne signal from the sample, exciting a vibrational mode of the sample and obtaining a spectrum of the excited sample.
11. A spectroscopy apparatus comprising a first excitation source arranged to excite a vibrational mode of a sample and a second excitation source arranged to excite an electronic mode of the sample, in which the mode is excited so as to obtain a spectrum in at least two dimensions.
12. A spectroscopy apparatus comprising an excitation source arranged to excite a vibrational mode of a sample, an excitation source arranged to excite a spectral portion surrounding a vibrational mode of a sample and a detector having a field of detection in at least one spatial dimension, in which the sample is excited so as to obtain a spectrum in at least two dimensions.
13. A method as claimed in any of claims 1 to 6 in which the parameter is controlled such that, where the emitted intensity is represented by I = (ELo)2 + (EHO)2 + (ELO X EHo) COS φ, E o is such that the cross term dominates EHO •
PCT/GB2004/004693 2003-11-07 2004-11-05 Method of spectroscopy WO2005047832A1 (en)

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EP04798417A EP1685370A1 (en) 2003-11-07 2004-11-05 Method of spectroscopy

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GBGB0326088.2A GB0326088D0 (en) 2003-11-07 2003-11-07 Method of spectroscopy

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Publication number Priority date Publication date Assignee Title
US10274310B2 (en) * 2016-12-22 2019-04-30 The Boeing Company Surface sensing systems and methods for imaging a scanned surface of a sample via sum-frequency vibrational spectroscopy
CN108896176B (en) * 2018-05-14 2019-10-11 浙江大学 A kind of Space Consistency bearing calibration of multi-optical spectrum imaging system

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ASBURY J B; STEINEL T; FAYER M D: "Using ultrafast infrared multidimensional correlation spectroscopy to aid in vibrational spectral peak assignments", CHEMICAL PHYSICS LETTERS, vol. 381, 20 October 2003 (2003-10-20), pages 139 - 146, XP002314684 *
M. CHO: "Ultrafast vibrational spectroscopy in condensed phases", PHYSCHEMCOMM, vol. 5, no. 7, 21 February 2002 (2002-02-21), pages 40 - 58, XP002314686 *
RUBTSOV I V; WANG J; HOCHSTRASSER R M: "Dual frequency 2D-IR spectroscopy heterodyned photon echo of the peptide bond", PNAS, vol. 100, no. 10, 13 May 2003 (2003-05-13), pages 5601 - 5606, XP002314685 *

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