1. Sergey Kucheryavski svk@bio.aau.dk Raman spectroscopy Acquisition, preprocessing and analysis of spectra

2. Raman spectrometer scheme Acquisition, preprocessing and analysis of Raman spectra2 Credits: http://www.doitpoms.ac.uk/tlplib/raman/method.php

3. Raman spectrometer Acquisition, preprocessing and analysis of Raman spectra3

4. Raman spectrometer Acquisition, preprocessing and analysis of Raman spectra4

5. Probes and fibers Acquisition, preprocessing and analysis of Raman spectra5

6. Non contact probe Acquisition, preprocessing and analysis of Raman spectra6

7. Acquisition of Raman spectra Acquisition, preprocessing and analysis of Raman spectra7

8. Raman signal is weak Only around 1 in every 30 million photons is Raman scattered

9. Acquisition of Raman spectra Acquisition, preprocessing and analysis of Raman spectra9 Issues Cosmic rays Noise Detection limits Fluorescence Parameters Laser frequency Laser power Exposure time Number of scans Preprocessing Spectral truncation Noise reduction Baseline correction Derivatives

10. Preprocessing Preprocessing – a way to improve signal for further analysis What can be improved –Noise reduction –Correction of baseline –Resolving merged meaks –Removing physical effects How it works: –X’ = F(X) –x ij = f j (x ij ) 106. Data preprocessing

11. Cosmic spikes Noise and detection limits Fluorescence and background correction

12. Cosmic spikes occasionally appears in spectra as very narrow peaks caused by high energy cosmic rays typical issue for CCD based instruments most of the acquisition software include algorithms to remove the effect Acquisition, preprocessing and analysis of Raman spectra12 Credits: Confocal Raman Microscopy. ed. Thomas Dieing, et al.

13. Cosmic spikes Noise and detection limits Fluorescence and background correction

14. Noise and detection limits CCD detectors have photon noise, dark noise and read noise Raman signal is weak To get a good signal/noise ratio cool CCD higher concentration longer exposure time more scans for the same sample de-noising preprocessing Acquisition, preprocessing and analysis of Raman spectra14

15. Noise and detection limits CCD detectors have photon noise, dark noise and read noise Raman signal is weak To get a good signal/noise ratio cool CCD higher concentration longer exposure time more scans for the same sample de-noising preprocessing Acquisition, preprocessing and analysis of Raman spectra15

16. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra16 25% ethanol t = 5s t = 3s t = 1s

17. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra17 10% ethanol t = 3s t = 1s

18. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra18 10% ethanol t = 3s t = 1s

19. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra19 1% ethanol t = 5s t = 3s t = 1s

20. Trancating spectra Acquisition, preprocessing and analysis of Raman spectra20 1% ethanol t = 5s t = 3s t = 1s

21. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra21 1% ethanol t = 5s t = 3s t = 1s

22. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra22 Butter t = 1s, 5 scans t = 1s, 3 scans t = 1s, 1 scan

23. Acquisition parameters and concentration Acquisition, preprocessing and analysis of Raman spectra23 Butter t = 1s, 5 scans t = 1s, 3 scans t = 1s, 1 scan

24. Playing with acquisition parameters Acquisition, preprocessing and analysis of Raman spectra24 Butter t = 3s, 5 scans t = 1s, 1 scan

25. Using filters for noise removal Linear filters: moving average, gaussian Wavelet decomposition Savitzky-Golay smoothing Acquisition, preprocessing and analysis of Raman spectra25 w = 5 d = 1

26. Using filters for noise removal Acquisition, preprocessing and analysis of Raman spectra26 SG filtered noised original

27. Cosmic spikes Noise and detection limits Fluorescence and background correction

28. Fluorescence Mechanism appears if molecules can absorb the laser radiation at particular wavelength the absorbed light excites electrons to higher energy levels electrons return to the ground state by emitting light of longer wavelength Acquisition, preprocessing and analysis of Raman spectra28

29. Fluorescence How decrease/get rid of fluorescence: remove impurities from solid samples using microprobes or confocal Raman microscopy (for solid samples) using lasers with wavelength in NIR range proper preprocessing (baseline correction) Acquisition, preprocessing and analysis of Raman spectra29 Features very common for colored (especially dark) samples several orders of magnitude stronger than Raman scattering has a broad emission

30. Fluorescence Color of samples Acquisition, preprocessing and analysis of Raman spectra30

31. Laser wavelength Visible — higher energy, stronger signal, deeper penetration, better resolution, fluorescence (good for inorganic materials) NIR — lower energy, weaker signal, worse resolution, smaller fluorescence effect (suitable for organic materials) Acquisition, preprocessing and analysis of Raman spectra31 Credits: http://www.horiba,com

32. Baseline correction Acquisition, preprocessing and analysis of Raman spectra32 Baseline shift and curvature caused by noise, fluorescence, CCD background, interference, etc. Automatic baseline correction

33. Baseline correction Acquisition, preprocessing and analysis of Raman spectra33 Automatic baseline correction d = 4

34. Baseline correction Acquisition, preprocessing and analysis of Raman spectra34 Automatic baseline correction d = 6

35. Baseline correction Acquisition, preprocessing and analysis of Raman spectra35 Semi-automatic baseline correction

36. Conclusions Issues Cosmic rays Noise Detection limits Fluorescence Acquisition, preprocessing and analysis of Raman spectra36 Parameters Laser frequency Laser power Exposure time Number of scans Preprocessing Spectral truncation Noise reduction Baseline correction Derivatives