Sergey Kucheryavski
svk@bio.aau.dk
Raman spectroscopy
Acquisition, preprocessing and analysis of spectra
Raman spectrometer scheme
Credits: http://www.doitpoms.ac.uk/tlplib/raman/method.php
Acquisition, preprocessing and analysis of Raman spectra
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Raman spectrometer
Acquisition, preprocessing and analysis of Raman spectra
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Raman spectrometer
Acquisition, preprocessing and analysis of Raman spectra
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Probes and fibers
Acquisition, preprocessing and analysis of Raman spectra
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Non contact probe
Acquisition, preprocessing and analysis of Raman spectra
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Acquisition of Raman spectra
Acquisition, preprocessing and analysis of Raman spectra
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Raman signal is weak
Only around 1 in every 30 million photons is Raman scattered
Acquisition of Raman spectra
Issues
Parameters
Preprocessing
•
Cosmic rays
•
Laser frequency
•
Spectral truncation
•
Noise
•
Laser power
•
Noise reduction
•
Detection limits
•
Exposure time
•
Baseline correction
•
Fluorescence
•
Number of scans
•
Derivatives
Acquisition, preprocessing and analysis of Raman spectra
9
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)
6. Data preprocessing
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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
Credits: Confocal Raman Microscopy. ed. Thomas Dieing, et al.
Acquisition, preprocessing and analysis of Raman spectra
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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
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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
25% ethanol
t = 5s
t = 3s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
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Acquisition parameters and concentration
10% ethanol
t = 3s
t = 1s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
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Acquisition parameters and concentration
10% ethanol
t = 3s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
18
Acquisition parameters and concentration
1% ethanol
t = 5s
t = 3s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
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Trancating spectra
1% ethanol
t = 5s
t = 3s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
20
Acquisition parameters and concentration
1% ethanol
t = 5s
t = 3s
t = 1s
Acquisition, preprocessing and analysis of Raman spectra
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Acquisition parameters and concentration
Butter
t = 1s, 5 scans
t = 1s, 3 scans
t = 1s, 1 scan
Acquisition, preprocessing and analysis of Raman spectra
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Acquisition parameters and concentration
Butter
t = 1s, 5 scans
t = 1s, 3 scans
t = 1s, 1 scan
Acquisition, preprocessing and analysis of Raman spectra
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Playing with acquisition parameters
Butter
t = 3s, 5 scans
t = 1s, 1 scan
Acquisition, preprocessing and analysis of Raman spectra
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Using filters for noise removal
• Linear filters: moving average, gaussian
• Wavelet decomposition
• Savitzky-Golay smoothing
1100
1000
900
1100
1000
w=5
800
800
700
700
600
600
500
500
400
1150
1155
d=1
900
1160
1165
1170
1175
1180
1185
1190
1195
1200
400
1150
1155
1160
1165
1170
1175
1180
1185
1190
1195
1200
1100
1000
900
800
700
600
500
400
1150
1155
1160
Acquisition, preprocessing and analysis of Raman spectra
1165
1170
1175
1180
1185
1190
1195
1200
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Using filters for noise removal
SG filtered
noised
original
Acquisition, preprocessing and analysis of Raman spectra
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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
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Fluorescence
Features
•
very common for colored (especially dark) samples
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several orders of magnitude stronger than Raman scattering
•
has a broad emission
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
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Fluorescence
Color of samples
Acquisition, preprocessing and analysis of Raman spectra
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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)
Credits: http://www.horiba,com
Acquisition, preprocessing and analysis of Raman spectra
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Baseline correction
Baseline shift and curvature caused by noise, fluorescence, CCD
background, interference, etc.
Automatic baseline correction
Acquisition, preprocessing and analysis of Raman spectra
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Baseline correction
Automatic baseline correction
d=4
Acquisition, preprocessing and analysis of Raman spectra
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Baseline correction
Automatic baseline correction
d=6
Acquisition, preprocessing and analysis of Raman spectra
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Baseline correction
Semi-automatic baseline correction
Acquisition, preprocessing and analysis of Raman spectra
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Conclusions
Issues
Parameters
Preprocessing
• Cosmic rays
• Laser frequency
• Spectral truncation
• Noise
• Laser power
• Noise reduction
• Detection limits
• Exposure time
• Baseline correction
• Fluorescence
• Number of scans
• Derivatives
Acquisition, preprocessing and analysis of Raman spectra
36