chemical analysis of complex biological systems …...kumar, v., et al. analyst, 4584-4593, 2015...
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SCHOOL OF ANALYTICALSCIENCES ADLERSHOF (SALSA)Humboldt-Universität zu Berlin · Office: Albert-Einstein-Str. 5–9, Adlershof
Unter den Linden 6 · 10099 Berlin · Germany
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Shirin M. Usmani
Victor Rodriguez
November 15th, 2016
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
OUTLINE
A. RAMAN SPECTROSCOPY
B. CAROTENOIDS
C. RAMAN MICROSCOPY
D. PHOTOBLEACHING
E. POLARIZATION
F. MAPPING
G. OTHER METHODS
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Butler H.J., et al. Nature Protocols, 664-687, 2016
PRINCIPLE OF INELASTIC AND ELASTIC SCATTERING
- Low probability event- 1 in 108 photons inelastically scattered- Insensitive technique
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Butler H.J., et al. Nature Protocols, 664-687, 2016
PRINCIPLE OF INELASTIC AND ELASTIC SCATTERING
Li and Church, Journal of Food and Drug Analysis, 29-48, 2014
TECHNOLOGICAL ADVANCES
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
- Efficient laser sources : Diode lasers, gas-based lasers, pulsed or continuous-wave
- Low-noise detectors : Charge-coupled devices (CCDs), electron-multiplying CCDs
- Effective Rayleigh filters : Dielectric edge filters, notch filters, Single or multistage monochromators
- High-throughput optics
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
RAMAN SPECTROSCOPIC MICROSCOPE SYSTEM
Butler H.J, et al. Nature Protocols, 664-687, 2016
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
CAROTENOIDS
Amorin-Carrilho, K.T., et al, Trends in Analytical Chemistry, 49-73, 2014
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
CAROTENOIDS
Carotenes Xanthophylls
Schulte, F., et al, Anal. Chem., 8426-8433, 2009
Amorin-Carrilho, K.T., et al, Trends in Analytical Chemistry, 49-73, 2014
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
FT-RAMAN SPECTRA OF PURE CAROTENOIDS STANDARDS
C=C C—C
- CH3
Schulz, H., et al. Biopolymers, 212-221, 2005
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
CAROTENOIDS ANALYSIS: IN-SITU
Schulz, H., et al. Biopolymers, 212-221, 2005
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
FLUORESCENCE
- Sample dependent
- Use of near-IR source
- 4 picosecond optical Kerr shutter
Gierlinger and Schwanninger, Spectroscopy, 69-89, 2007
Fu, X., et al. Food Anal. Methods, 2501–2508, 2016
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
C=C :1525 cm-1
MICROSCOPY + RAMAN SPECTROSCOPY = NON-DESTRUCTIVE ANALYSIS
Schulz, H., et al. Biopolymers, 212-221, 2005
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
RESOLUTION: SPATIAL
C=C :1525 cm-1
∆x = 0.61λ/NA
Gierlinger and Schwanninger, Spectroscopy, 69-89, 2007
Schulz, H., et al. Biopolymers, 212-221, 2005
- shorter wavelength and high-magnification optics
Spectral Resolution
- Higher excitation wavelength
Depends on the scientific question:- molecular informationOR - localized information
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
PENETRATION DEPTH
Butler H.J, et al. Nature Protocols, 664-687, 2016
- Shorter wavelength, higher energy but more scattered- Hence, ideal for studying the surface of a tomato sample- But, need longer wavelength if information needed about the carotenoid
composition of a tomato core
PHOTOBLEACHING
Scholtes-Timmerman, M., Analyst, 387–393. 2009
• Changes in the electronic structure
• Important for samples containing
carotenoids
• Carotenoids signal steadily
decreases.
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
PHOTOBLEACHING
• The variance affects Reproducibility
• Orders of magnitude stronger
Kumar, V., et al. Analyst, 4584-4593, 2015
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Post-acquisition data treatment:
• Omitting carotenoid spectral regions
Most variation in 1157cm −1 and 1525cm −1
DEALING WITH COMPOUNDS SUSCEPTIBLE TO PHOTOBLEACHING :
Scholtes-Timmerman, M., Analyst, 387–393. 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Elimination of these peaks does
not eliminate variation
The width of the carotenoid bands
force to discard other spectral
features hidden beneath.
DEALING WITH COMPOUNDS SUSCEPTIBLE TO PHOTOBLEACHING :
Kumar, V., et al. Analyst, 4584-4593, 2015
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
DEALING WITH COMPOUNDS SUSCEPTIBLE TO PHOTOBLEACHING :
Spectra processing:
EMSC: backgroundsignal correction usingExtended MultiplicativeScatter Correction
EMSC-SIS : ExtendedMultiplicative ScatterCorrection and SpectralInterference Subtraction
Scholtes-Timmerman, M., Analyst, 387–393. 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
DEALING WITH COMPOUNDS SUSCEPTIBLE TO PHOTOBLEACHING :
Photodecomposition:
• Before data acquisition
• Long time 30-60 min
• a) 10min• b) 30min• c) 50min• d) 70min
Schulte, F., et al. Analytical Chemistry, 8426–8433, 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
The difference spectra can be used to study the carotenoids and their interactions with the biological matrix
Schulte, F., et al. Analytical Chemistry, 8426–8433, 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
POLARIZATION:
The Raman spectrum depends on
the orientation and polarization of
light.
Intensities vary depending on the
angle between polarizability tensor
of a specific molecular vibration
and the exciting source.
N. Gierlinger, M. Schwanninger, Plant Physiol. 1246-54. 2006
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Cross-section of wood L. Procera
N. Gierlinger, M. Schwanninger, Plant Physiol. 1246-54. 2006
Polarized Raman spectroscopy: information about structure and orientation
B: cellulose parallel along the fiber from 2774 to 3026cm −1
C: cellulose oriented with a high angle inrespect to the fiber from 1067 to 1106cm −1
(orientation-sensitive cellulose band 1097cm −1)
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
MAPPING:
Pre-processing (minimizing variability):
Cosmic rays
Fluorescence
Poor signal to noise ratio
Baseline correction (polynomial fitting, derivative spectra)
Truncating the spectra
Feature extraction: PLS or PCA
Classification: HCA or PCA
Large data sets that require computational processing
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
A better understanding on structure, chemical composition of plant cells, tissues and organs.
Baranska, M. et al. Current Analytical Chemistry, 108–127, 2013
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Raman spectrum is a combination of the spectra of the single compounds.
Schulte, F., Journal of Biophotonics, 542–547, 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
• HPTLC/AMD
• HPLC is the most used method for quantification, but carotenoids need to be
extracted (destructive)
OTHER METHODS:
Schulte, F., et al. Analytical Chemistry, 8426–8433, 2009
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
Sample preparation
Choose instrumentation
Resonant vs non resonant
Photobleaching
Polarization
Mapping
Supporting methods
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy
SUMMARY:
Thank you for your attention!
Chemical Analysis of Complex Biological Systems by Raman Spectroscopy