sergey kucheryavski svk@bio.aau.dk raman spectroscopy acquisition, preprocessing and analysis of...

Sergey Kucheryavski

svk@bio.aau.dk

Raman spectroscopy

Acquisition, preprocessing and analysis of spectra

Raman spectrometer scheme

Acquisition, preprocessing and analysis of Raman spectra 2

Credits: http://www.doitpoms.ac.uk/tlplib/raman/method.php

Raman spectrometer

Acquisition, preprocessing and analysis of Raman spectra 3

Raman spectrometer

Acquisition, preprocessing and analysis of Raman spectra 4

Probes and fibers

Acquisition, preprocessing and analysis of Raman spectra 5

Non contact probe

Acquisition, preprocessing and analysis of Raman spectra 6

Acquisition of Raman spectra

Acquisition, preprocessing and analysis of Raman spectra 7

Raman signal is weak

Only around 1 in every 30 million photons is Raman scattered

Acquisition of Raman spectra

Acquisition, preprocessing and analysis of Raman spectra 9

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

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)

– xij = fj(xij)

106. Data preprocessing

Cosmic spikes

Noise and detection limits

Fluorescence and background correction

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 spectra 12

Credits: Confocal Raman Microscopy. ed. Thomas Dieing, et al.

Cosmic spikes

Noise and detection limits

Fluorescence and background correction

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 spectra 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 spectra 15

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 16

25% ethanol

t = 5s

t = 3s

t = 1s

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 17

10% ethanol

t = 3s

t = 1s

t = 1s

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 18

10% ethanol

t = 3s

t = 1s

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 19

1% ethanol

t = 5s

t = 3s

t = 1s

Trancating spectra

Acquisition, preprocessing and analysis of Raman spectra 20

1% ethanol

t = 5s

t = 3s

t = 1s

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 21

1% ethanol

t = 5s

t = 3s

t = 1s

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 22

Butter

t = 1s, 5 scans

t = 1s, 3 scans

t = 1s, 1 scan

Acquisition parameters and concentration

Acquisition, preprocessing and analysis of Raman spectra 23

Butter

t = 1s, 5 scans

t = 1s, 3 scans

t = 1s, 1 scan

Playing with acquisition parameters

Acquisition, preprocessing and analysis of Raman spectra 24

Butter

t = 3s, 5 scans

t = 1s, 1 scan

Using filters for noise removal

• Linear filters: moving average, gaussian

• Wavelet decomposition

• Savitzky-Golay smoothing

Acquisition, preprocessing and analysis of Raman spectra 25

w = 5 d = 1

Using filters for noise removal

Acquisition, preprocessing and analysis of Raman spectra 26

SG filtered

noised

original

Cosmic spikes

Noise and detection limits

Fluorescence and background correction

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 spectra 28

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 spectra 29

Features

• very common for colored (especially dark) samples

• several orders of magnitude stronger than Raman scattering

• has a broad emission

Fluorescence

Color of samples

Acquisition, preprocessing and analysis of Raman spectra 30

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 spectra 31

Credits: http://www.horiba,com

Baseline correction

Acquisition, preprocessing and analysis of Raman spectra 32

Baseline shift and curvature caused by noise, fluorescence, CCD

background, interference, etc.

Automatic baseline correction

Baseline correction

Acquisition, preprocessing and analysis of Raman spectra 33

Automatic baseline correction

d = 4

Baseline correction

Acquisition, preprocessing and analysis of Raman spectra 34

Automatic baseline correction

d = 6

Baseline correction

Acquisition, preprocessing and analysis of Raman spectra 35

Semi-automatic baseline correction

Conclusions

Issues

• Cosmic rays 

• Noise

• Detection limits

• Fluorescence

Acquisition, preprocessing and analysis of Raman spectra 36

Parameters

• Laser frequency 

• Laser power

• Exposure time

• Number of scans 

Preprocessing

• Spectral truncation

• Noise reduction 

• Baseline correction

• Derivatives