Chemical Analysis on complex biological systems by Raman spectroscopy. Janina Kneipp
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1 Chemical Analysis on complex biological systems by Raman spectroscopy Janina Kneipp Analytical Strategies J. Kneipp Nov
2 Raman scattering and resonant Raman scattering S 1 resonant Raman scattering S 1 h Laser h anti Stokes h Stokes h Laser h Molecule v 1 v 0 S 0 h Stokes h Molecule v 1 v 0 S 0 Stokes Laser Molecule anti Stokes Laser + Molecule Analytical Strategies J. Kneipp Nov
3 Cross sections considered Fluorescence IR absorption abs cm Fluorescence FL cm 2 Raman 1174 nonresonant resonant Stokes Shift [cm ] 730 Raman scattering RS cm 2 Analytical Strategies J. Kneipp Nov
4 Raman shift with respect to ex = 488 nm Raman shift (cm 1 ) Analytical Strategies J. Kneipp Nov
5 Raman scattering: classical picture Structure of the dipole moment that is induced by the incoming field Rayleigh Anti Stokes Stokes Analytical Strategies, preparation lecture WS2014/15
6 Dispersion by grating From R.L. McCreery Raman Spectroscopy for Chemical Analysis Wiley (2000) Linearity with respect to wavelength, not to Raman shift! Analytical Strategies J. Kneipp Nov
7 Dispersive Set up From R.L. McCreery Raman Spectroscopy for Chemical Analysis Wiley (2000) Analytical Strategies J. Kneipp Nov
8 Grating determines spectral range and resolution From R.L. McCreery Raman Spectroscopy for Chemical Analysis Wiley (2000) Analytical Strategies J. Kneipp Nov
9 Example Raman setup (180 backscattering geometry) NIR Laser at 830 nm IF LP (O) N PA (O) S FL Spectrograph Laser Path CCD Notch Filter: Elimininate Rayleigh scattered light Raman path Obj From Lin Ping Choo Smith et al. in Biomedical Vibrational Spectroscopy P. Lasch & J. Kneipp (eds.), Wiley 2008 Analytical Strategies J. Kneipp Nov
10 Quantum yield of CCD Detektor (example) From R.L. McCreery Raman Spectroscopy for Chemical Analysis Wiley (2000) Analytical Strategies J. Kneipp Nov
11 Lifetimes and time resolved detection el~ ns 1 k It is possible to separate Raman from fluorescence photons using a gate! Analytical Strategies J. Kneipp Nov
12 What is a good excitation wavelength? fluorescence pre resonant RS normal RS resonant RS From R.L. McCreery Raman Spectroscopy for Chemical Analysis Wiley (2000) Analytical Strategies J. Kneipp Nov
13 Correlating spectral with spatial information: Microspectroscopic Mapping and Imaging (1) Microspectroscopic mapping (2) Spectral analysis Sample: tissue, cell etc Raman shift /cm -1 Single spectral parameters intensities, ratios, band correlations vibrational frequency & shifts Multivariate information Spectrum = vector, pattern Principal components, cluster analysis, artificial neural networks (3) Molecular Image: Combination of spectral parameters and spatial coordinates H&E unstained Raman image (e.g. K means cluster) Analytical Strategies J. Kneipp Nov
14 Lignin and cellulose distribution Agarwal, U. P. (2006). Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana). Planta, 224(5), Analytical Strategies J. Kneipp Nov
15 Lignin and cellulose spectra Agarwal, U. P. (2006) Planta, 224(5), Sarkar, P.. (2009) Journal of Experimental Botany, 60, Analytical Strategies J. Kneipp Nov
16 Lignin staining Raman Richter, S., Mussig, J., & Gierlinger, N. (2011). Functional plant cell wall design revealed by the Raman imaging approach. Planta, 233(4), Analytical Strategies J. Kneipp Nov
17 Polarization dependence of Raman scattering Benzol stark polarisierte Banden Depolarisationsgrad I I Analytical Strategies J. Kneipp Nov
18 Information on cellulose orientation Gierlinger, N., Luss, S., Konig, C., Konnerth, J., Eder, M., & Fratzl, P. (2010). Cellulose microfibril orientation of Picea abies and its variability at the micron level determined by Raman imaging. Journal of Experimental Botany, 61(2), Polarization direction of incident laser increases in angle with respect to the micofibril orientation Analytical Strategies J. Kneipp Nov
19 Lignin signals are not sensitive to polarization Lignin signals Cellulose S1 layer with high microfibril angle Gierlinger, N., Luss, S., Konig, C., Konnerth, J., Eder, M., & Fratzl, P. (2010). Cellulose microfibril orientation of Picea abies and its variability at the micron level determined by Raman imaging. Journal of Experimental Botany, 61(2), Analytical Strategies J. Kneipp Nov
20 Using spectral fingerprints for imaging stained cell unstained cell map of cluster analysis result frequency map (amide I) Analytical Strategies J. Kneipp Nov J. Kneipp et al. Biochim Biophys Acta, 2003, 1639:
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