MULTI-EXCITATION RAMAN SPECTROSCOPY METHOD AND APPARATUS

20250052683

18/717742

December 07, 2022

There is provided a method of identifying one or more substances in a sample by: illuminating the sample with light of each of a plurality of different excitation modes; measuring an intensity and/or polarisation of light from the sample at a plurality of wavelengths to obtain a measured spectrum for each of the excitation modes; and identifying one or more substances in the sample using the measured spectra together, wherein: the excitation modes differ in one or both of wavelength and polarisation; and the identifying of the one or more substances uses contributions to the measured spectra from a plurality of photophysical processes in the sample including inelastic scattering of light. An apparatus suitable for carrying out the method is also provided.

UNIVERSITY OF SOUTHAMPTON (Southampton Hampshire, GB)

1. A method of identifying one or more substances in a sample comprising: illuminating the sample with light of each of a plurality of different excitation modes; measuring an intensity and/or polarisation of light from the sample at a plurality of wavelengths to obtain a measured spectrum for each of the excitation modes; and identifying one or more substances in the sample using the measured spectra together, wherein: the excitation modes differ in one or both of wavelength and polarisation; and the identifying of the one or more substances uses contributions to the measured spectra from a plurality of photophysical processes in the sample including inelastic scattering of light.

2. The method of claim 1, wherein identifying the one or more substances comprises using a multivariate analysis, for example principal component analysis or linear discriminant analysis.

3. (canceled)

4. The method of claim 2, wherein identifying the one or more substances comprises combining the measured spectra using the multivariate analysis to obtain a multi-dimensional signature of the sample.

5. The method of claim 4, wherein identifying the one or more substances further comprises comparing the multi-dimensional signature to one or more reference signatures.

6. The method of claim 1, wherein identifying the one or more substances comprises using a machine-learning algorithm, for example a support vector machine or a neural network.

7. (canceled)

8. The method of claim 1, wherein identifying the one or more substances comprises classifying the one or more substances.

9. The method of claim 1, wherein identifying the one or more substances does not comprise processing to reduce the contribution to the measured spectra of any photophysical processes in the sample.

10. The method of claim 1, wherein the plurality of photophysical processes in the sample further includes one or more of fluorescence, photoluminescence, phosphorescence, elastic scattering, and reflection.

11. The method of claim 1, wherein the plurality of photophysical processes in the sample further includes fluorescence, and identifying the one or more substances does not comprise processing to reduce the contribution to the measured spectra of the fluorescence in the sample.

12. The method of claim 1, wherein the inelastic scattering of light comprises Raman scattering.

13. The method of claim 1, wherein the polarisation of at least one of the excitation modes comprises linear polarisation, circular polarisation, or elliptical polarisation.

14. The method of wherein two or more of the excitation modes differ from one another in polarisation.

15. The method of claim 1, wherein the wavelengths of the excitation modes comprise one or more visible light wavelengths and/or one or more infra-red light wavelengths.

16. The method of claim 15, wherein the one or more visible light wavelengths comprise a wavelength in the range 400-700 nm, and the one or more infra-red light wavelengths comprise a wavelength in the range 700-3000 nm.

17. The method of claim 15, wherein the wavelengths of the excitation modes comprise one or more of 405 nm, 532 nm, 633 nm, 785 nm, and 1064 nm.

18. The method of claim 1, wherein two or more of the excitation modes differ from one another in wavelength, for example by at least 20 nm.

19. (canceled)

20. The method of claim 18, wherein the wavelengths of the excitation modes are such that the measured spectra comprise two or more of a non-resonant Raman spectrum, a pre-resonant Raman spectrum, and a resonant Raman spectrum.

21. The method of claim 1, wherein illuminating the sample with light of each of a plurality of different excitation modes comprises either a) illuminating the sample simultaneously with light of each of the excitation modes, or b) illuminating the sample sequentially with light of each of the excitation modes.

22. (canceled)

23. The method of claim 1, wherein measuring an intensity and/or polarisation of light from the sample for each of the excitation modes comprises filtering out light at the wavelength of the excitation mode.

24. The method of claim 1, wherein the one or more substances comprise microorganisms, for example bacteria or archaea, and identifying the one or more substances comprises classifying the microorganisms.

25. The method of claim 24, wherein classifying the microorganisms comprises identifying a category of the microorganisms, wherein the category comprises one or more of a taxonomic group of the microorganisms, for example a sub-species, strain, species, genus, family, order, or class, and a phenotype of the microorganisms, for example a type of anti-microbial resistance or anti-biotic susceptibility.

26. (canceled)

27. The method of claim 25, wherein the sample comprises microorganisms of two or more categories, and identifying the one or more substances comprises determining a quantity and/or relative proportion of microorganisms of each of the two or more categories.

28. The method of claim 1, wherein the one or more substances comprise any type of amyloid fibrils, amyloid plaques or its precursors, tau and phospho-tau, huntingtin, other markers of proteinopathies, or extracellular matrix components, for example collagen and/or elastin.

29. (canceled)

30. The method of claim 1, wherein the sample is a biological sample.

31. The method of claim 30, wherein the sample is obtained from an organism, and the method further comprises determining a type, a likelihood, a severity, and/or a stage of a disease or condition for the organism on the basis of the identification of the one or more substances, optionally wherein the organism is a human.

32. The method of claim 1, wherein the sample is obtained from a non-biological source.

33. The method of claim 32, wherein the method further comprises determining a likelihood or severity of contamination of the non-biological sourced, for example a biological contamination, on the basis of the identification of the one or more substances.

34. (canceled)

35. An apparatus for identifying one or more substances in a sample comprising: an illumination source configured to illuminate the sample with light of each of a plurality of different excitation modes; a detector configured to measure an intensity and/or polarisation of light from the sample at a plurality of wavelengths to obtain a measured spectrum for each of the excitation modes; and a processing unit configured to identify one or more substances in the sample using the measured spectra together, wherein: the excitation modes differ in one or both of wavelength and polarisation; and the processing unit is configured to identify the one or more substances using contributions to the measured spectra from a plurality of photophysical processes in the sample including inelastic scattering of light.

36. The apparatus of claim 35, wherein the illumination source comprises a wavelength-tuneable illumination source.

37. The apparatus of claim 35, wherein the illumination source comprises a plurality of sub-sources, each sub-source configured to emit light at a different wavelength from the other sub-sources.

38. The apparatus of claim 35, wherein the illumination source is configured to emit polarised light.

39. The apparatus of claim 38, wherein the polarisation of the light emitted by the illumination source is tuneable.

40. The apparatus of claim 38, wherein the illumination source comprises a plurality of sub-sources, each sub-source configured to emit light having a different polarisation from the other sub-sources.

41. The apparatus of claim 35, wherein the bandwidth of light emitted by the illumination source is sufficiently narrow to resolve a Raman linewidth of 50 cm−1 or less.

42. The apparatus of claim 35, wherein either a) the detector is configured to detect light reflected and/or backscattered from the sample, or b the detector is configured to detect light transmitted through and/or scattered by the sample.

43. (canceled)

44. The apparatus of claim 35, wherein the apparatus further comprises one or more of: a) a filtering element, for example a shortpass optical filter, longpass optical filter, notch optical filter, bandpass optical filter, or electro-optical modulator, the filtering element configured to remove light at the wavelength of the excitation mode from the light from the sample; b) a detection polariser configured to select light having a predetermined polarisation from the light from the sample; and c) a resolving element, for example a spectrograph, a grating, a prism, or an interferometer, the resolving element configured to spectrally resolve the light from the sample.

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

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edspap.20250052683