Raman Spectroscopy for the analysis of plant cell wall

Biochemical composition and Si detection on cucumber roots and stem

18.11.2014Oleksandr Zavoiura, Anna Lopez de Guerenu and Bárbara Gonzalez

Raman Spectroscopy

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• Measurement of inelastic scattering

• Requirement for activity:

• Interfering factor: Fluorescence

1

1

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Raman Spectroscopy2

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Raman Spectroscopy

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Plants cell wall

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Cucumber Plant Illustration. 4 Cucumber Cell5. ≈100μm Cucumber Cell Wall6.

• The cell walls is essential for the plant7: Shape – Support – Growth– Signalling – Transport.

• The main components are: Proteins–Polysaccharides– Lipids – Aromatic substances.

Silicic acid and Silicification

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Study of silica deposition in cucumber (cucumis sativus from cucurbitaceae family) plant cell walls

• Which organisms? Plants, diatoms (algae) and sponges.

• Silica nutrition benefits? Plant defence, stress resistance and growth factors.

• Mechanisms? Objective of study in plant physiology.

pKa=9.8

Scheme of the Silica up-taking reactions.8

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Raman Spectroscopy procedure

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Fresh plant sample give the best results

• Planar surface and intact cell walls

• Be aware of stem and fiber orientation in order to cut and

investigate defined planes

• Thickness 8–20 μm

• Bigger than focal depth of laser beam (To avoid

signal from the coverslip)

• High expertise to cut by hand Rotatory microtome

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Sample preparation: Cutting considerations

Rotatory Microtome semi-automaticFrom Mikron Instruments Inc 10

Cryosectioning

• combination of microtome and cryostat

• snapfreezing (liquid N2)

• thickness between 10 and 30 μm

• timing: 1 h to 1 d

Embedding

• PEG works the best no matrix effect

• Resin not as good noise is increased (especially at λ = 532 nm)

• timing: 1 week

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Sample preparation: cryostat vs. embedding

Cryostat for cryosectioningCM1800 from Leica 11

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Raman Spectroscopy procedure

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Laser sources

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488 nmAr+

532 nmNd:YAG

633 nmHe-Ne

785 nmGaAlAs

400 nm 500 nm 600 nm 700 nm

UV IR

High Raman Intensity Low

Good Spatial resolution Poor

High Fluorescence Low

Inorganic SamplesMapping

Resonant Raman ScatteringPreferred for Highly fluorescent compounds

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Confocal Raman Spectroscopy: setup

Big Raman Lab (TU Berlin)12

LaserSources

Output SpectraSALSA’s qualified PhD Student

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Confocal Raman Spectroscopy: setup

Spectrometer

Confocal Microscope

CCD-Detector

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Cucumber: What to expect

Scale model of a portion of a primary plant cell wall 13 Raman spectra of a cell wall membrane of onion (1),carrot (2), polypogon (3), rice (4) and sweet corn (5) 14

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Cucumber: What to expectStudy of silica deposition on cucumber roots and stem vs non-silica samples

Electron micrographs of silica structures from plants. Stomata of mature E. arvense surrounded by pilulae encrusted with rosettes; the observed specimens contain 0.1 % w/w C, with the remainder being silica; left, upper surface; right, lower surface. 8

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Cucumber: What to expectStudy of silica deposition on cucumber roots and stem vs non-silica samples

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Cell wall compounds I

Lignin16

Cellulose15

Pectic compounds17

Rhamnogalacturonan I

Homogalacturonan

Rhamnogalacturonan II

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Cell wall compounds I9

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Cell wall compounds II

Spectra of chlorophyll water aggregates, suspended in decane. Lower spec-trum: chlorophyll a, excitation 457.9 nm. Upper spectrum, chlorophyll b, excitation 488 nm. 18

Chlorophyll

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Raman wavenumber

[cm-1]Assignment

2938 Lignin (20)

1658 Lignin (20)

1650 Protein Amide I (22)

1602 Lignin (20)

1554 Chlorophyll (21)

1533 Chlorophyll(21)

1445 Lipids, Protein ᵟ(CH2, CH3)

1350 Chlorophyll(21)

1333 Lignin (20)

1292 Chlorophyll(21)

1268 Protein Amide III (22)

Cell wall compounds III19

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Polarization dependence

• For crystalline structures the intensity of the peak depends on the polarization of the light

E.g. cellulose peak at 370 cm-1

• By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall can be investigated

Hypothetical arrangement of organic layers in the algae (diatom) cell wall.

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Polarization dependence

Fourier transform-Raman spectra of wheat epidermis walls with parallel and perpendicular polarization: (A) stalk, (B) sheath, and (C) node. Raman Intensity is given on a relative scale. 24

Degree of polarisation (p):

y2= anisotropy

α= polarisationtensor

Symmetry=100% p=3/4; α=0

Symmetry<100% 0<p<3/4; α≠0

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Raman Spectroscopy procedure

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Data analysis I: Spectra processing

• Cosmic Ray Removal – to get rid of spikesfrom the CCD detector in the spectra.

• Smoothing algorithms – to reduce the noise inthe recorded spectra.

• Background–Baseline correction – to eliminate changes in background (suitable for all spectra in the data set)

• Additional parameters – depending on the goalof the data analysis

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• Univariate analysis – Single band imaging:

Each spectrum determines one value of the corresponding pixel in the image.

• Simple filters: a chemical image can be generated by focusing on an area of the map and integrating

over one specific peak of interest. Information about the spatial distribution.

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Data analysis II: Image generation

Optical image of the analysis of Tomato cell wall. A) Microscopic image B) polysaccharides at 2940cm-1 C) Pectin at 854cm-1 D) Cellulose at 1090cm-1, γ(COC)glycosidic.19

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Data analysis II: Image generationMultivariate analysis:

Hyperspectral images generated from matrices containing redundant information

• Principal Component Analysis (PCA): Math–Orthogonal linear transformation. Effective to reduce the data set to 5–15 principal components, plus the residual error. Contains all the relevant info of the image.

a) 2D PCA-scores plot of PC1 vs PC2 b) 1D PCA-loadings plot of PC1 vs Raman shift. Raman shift range 200 – 2000 cm-1.25

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PCA scores images and loading spectra. A) PC2 mainly Pectin B) PC3 mainly Cellulose C) PC2 and PC3 depicted as Pectin and Cellulose distribution D) PC2 loading E) PC3 loading. Analysis of Tomato cell walls.19

Data analysis II: Image generation

• Multivariate curve resolution (MCR):

• recovery of the response profiles of more than one component in unresolved and unknown mixtures

• provides information about the nature and composition of these mixtures.

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MCR concentration images and the spectra of the sampled pure-components: A) component 1; B) component 2; C) component 3. 19

A) Assigned mainly to Pectin compoundsCharacteristic peaks: 2949, 1751, 855 and 816 cm−1

B) Assigned mainly to Cellulose Characteristic peaks: 2895, 1378, 1122, 1093 and 971 cm−1

C) Assigned mainly to hemicelluloseCharacteristic peaks: 2933 and 1257 cm−1

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Advantages Disadvantages

• Can be used with solids and liquids• No sample preparation needed• Not interfered by water• Non-destructive• Highly specific like a chemical fingerprint of a material• Raman spectra are acquired quickly within seconds• Samples can be analyzed through glass or a polymer

packaging• Laser light and Raman scattered light can be

transmitted by optical fibers over long distances for remote analysis

• Raman spectra can be collected from a very small volume (< 1 µm in diameter)

• Inorganic materials are normally easier analyzed by Raman than by infrared spectroscopy

• Can not be used for metals or alloys• The Raman effect is very weak. The detection needs a

sensitive and highly optimized instrumentation.• Fluorescence of impurities or of the sample itself can

hide the Raman spectrum.• Sample heating through the intense laser radiation

destroy the sample or hide the Raman spectrum.

Methods to complement Raman results

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• SEM Scanning Electron Microscopy• Back scattering electrons • EDX + WDX

• WAXS / SAXS Wide/Small Angle X-Rays Scattering

• Polarized light

• Acoustic scanning microscopy

• ICP-MSInduced-Coupled Plasma MS

Stiffness and density of the sample

Microfibrils orientation / Microfibrils Angle (MFA)

Structural differences between the cell wall regions.

Study of MFA

Elemental composition of the sample.26

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References1- Raman Spectroscopy. Wikipedia. Online (http://en.wikipedia.org/wiki/Raman_spectroscopy), visited 12.11.2014.2- Raman.de Dr. Bernd Dippel. Online (www.raman.de), visited 16.11.2014.3- Fenn M. B. et al. Raman Spectroscopy for Clinical Oncology. Advance in Optical Technology, Hindawi Publishing Corporation, 2011. 4- East-west seed Plant Doctor. Online (http://plantdoctor.eastwestseed.com/plant-parts/Cucumber), visited 11.11.2014.5- Gottalovebio, Cells. Online (http://gottalovebio.wikispaces.com/Team+5), visited 11.11.2014.6- Yellow tang, Cell wall in plants. Online (http://www.yellowtang.org/images/cell_walls_in_plant_c_ph_784.jpg), visited 11.11.2014.7- College of St. Benedict/St. John's University, Biology Department, Plant Physiology. Online

(http://employees.csbsju.edu/ssaupe/biol327/Lecture/cell-wall.htm), visited 11.11.2014.8- Heater A. C. and Carole C. P. Silica in Plants: Biological, Biochemical and Chemical Studies. Annals of Botany 100; 1383-1389, 2007.9- Gierlinger N. et. al. Imaging of plant cell wall by confocal Raman microscopy. Nature Protocol, 7 no.9; 1694-1708, 2012.10- Mikron Instruments Inc. Online (www.mikronet.com), visited 11.11.2014.11- Leica BIOSYSTEMS. Online (www.leicabiosystems.com), visited 11.11.2014.12- Pictures acquired from Raman Laboratory from Prof. Peter Hildebrandt research group, TU Berlin. Taken 13.11.2014.13- Cosgrove D., Nature Reviews Molecular Cell Biology 6, 850-861, 2005.14- Sene et al. Plant Physiology. vol. 106 no. 4, 1623-1631, 1994.15- Sarkar P et al., J. of Exp. Botany, Vol. 60, No 13, 3615-3635, 2009.16- Wikipedia, Lignin. Online (http://en.wikipedia.org/wiki/Lignin), visited 13.11.2014.17- Ochoa-Villarreal M et al. Plant Cell Wall Polymers: Function, Structure and Biological Activity of Their Derivatives. Polymerization 4, 63-86, 2012. 18- M. lutz, Resonant Raman spectral of chlorophyll in solution. Journal of Raman Spectroscopy, Vol 2; 497-516, 1974.19- Chylinska M, et. al. Imaging of polysaccharides in the tomato cell wall with Raman microspectroscopy. Plant Methods 10:14, 2014.20- Rowel M.R. Handbook of Wood Chemistry and Wood Composites, Second Edition 2013, p. 233.21- Z.Cai, et al. Biochimica et Biophysica Acta (BBA) – Bioenergetics, Vol.1556,(2–3), 2002, 89-91.22- Schulte F. et al. , Anal. Chem., 2008, 80, 9551-9556.24- Cao et al, Ann Bot. Jun 2006; 97(6): 1091–1094.25- Mobili P. et. al. Multivariate analysis of Raman spectra applied to microbiology: Discrimination of microorganisms at the species level.

Revista mexicana de fisica 56 (5) 378–385, 2010.26- Sielbold M, et. al. Application of elemental bioimaging using laser ablation ICP-MS in forest pathology: distribution of elements in the bark of Picea sitchensis following wounding. Anal Bioanal Chem 402:3323– 3331, 2012.

Thank you …!

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Iona Sighiartau

Robert Harmel

Sven Schulz

Konrad Löhr

José Villatoro

Thi Hoa Hoang

Felix Rösicke

Virginia Merk

&

Prof. Janina Kneipp