sergey kucheryavski [email protected] raman spectroscopy acquisition, preprocessing and analysis of...
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Sergey Kucheryavski
Raman spectroscopy
Acquisition, preprocessing and analysis of spectra
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Raman spectrometer scheme
Acquisition, preprocessing and analysis of Raman spectra 2
Credits: http://www.doitpoms.ac.uk/tlplib/raman/method.php
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Raman spectrometer
Acquisition, preprocessing and analysis of Raman spectra 3
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Raman spectrometer
Acquisition, preprocessing and analysis of Raman spectra 4
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Probes and fibers
Acquisition, preprocessing and analysis of Raman spectra 5
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Non contact probe
Acquisition, preprocessing and analysis of Raman spectra 6
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Acquisition of Raman spectra
Acquisition, preprocessing and analysis of Raman spectra 7
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Raman signal is weak
Only around 1 in every 30 million photons is Raman scattered
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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
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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
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Cosmic spikes
Noise and detection limits
Fluorescence and background correction
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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.
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Cosmic spikes
Noise and detection limits
Fluorescence and background correction
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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 14
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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
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Acquisition parameters and concentration
Acquisition, preprocessing and analysis of Raman spectra 16
25% ethanol
t = 5s
t = 3s
t = 1s
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Acquisition parameters and concentration
Acquisition, preprocessing and analysis of Raman spectra 17
10% ethanol
t = 3s
t = 1s
t = 1s
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Acquisition parameters and concentration
Acquisition, preprocessing and analysis of Raman spectra 18
10% ethanol
t = 3s
t = 1s
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Acquisition parameters and concentration
Acquisition, preprocessing and analysis of Raman spectra 19
1% ethanol
t = 5s
t = 3s
t = 1s
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Trancating spectra
Acquisition, preprocessing and analysis of Raman spectra 20
1% ethanol
t = 5s
t = 3s
t = 1s
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Acquisition parameters and concentration
Acquisition, preprocessing and analysis of Raman spectra 21
1% ethanol
t = 5s
t = 3s
t = 1s
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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
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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
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Playing with acquisition parameters
Acquisition, preprocessing and analysis of Raman spectra 24
Butter
t = 3s, 5 scans
t = 1s, 1 scan
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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
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Using filters for noise removal
Acquisition, preprocessing and analysis of Raman spectra 26
SG filtered
noised
original
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Cosmic spikes
Noise and detection limits
Fluorescence and background correction
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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
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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
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Fluorescence
Color of samples
Acquisition, preprocessing and analysis of Raman spectra 30
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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)
Acquisition, preprocessing and analysis of Raman spectra 31
Credits: http://www.horiba,com
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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
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Baseline correction
Acquisition, preprocessing and analysis of Raman spectra 33
Automatic baseline correction
d = 4
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Baseline correction
Acquisition, preprocessing and analysis of Raman spectra 34
Automatic baseline correction
d = 6
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Baseline correction
Acquisition, preprocessing and analysis of Raman spectra 35
Semi-automatic baseline correction
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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