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Imaging of plant cell walls by confocal Raman microscopy

Nature Protocols volume 7, pages 1694–1708 (2012)Cite this article

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Abstract

Raman imaging of plant cell walls represents a nondestructive technique that can provide insights into chemical composition in context with structure at the micrometer level (<0.5 μm). The major steps of the experimental procedure are described: sample preparation (embedding and microcutting), setting the mapping parameters, and finally the calculation of chemical images on the basis of the acquired Raman spectra. Every Raman image is based on thousands of spectra, each being a spatially resolved molecular 'fingerprint' of the cell wall. Multiple components are analyzed within the native cell walls, and insights into polymer composition as well as the orientation of the cellulose microfibrils can be gained. The most labor-intensive step of this process is often the sample preparation, as the imaging approach requires a flat surface of the plant tissue with intact cell walls. After finishing the map (acquisition time is ∼10 min to 10 h, depending on the size of the region of interest and scanning parameters), many possibilities exist for the analysis of spectral data and image generation.

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Figure 1: Raman spectra acquired from a Ramie fiber.
Figure 2: Typical spectra for lignin, pectin and cellulose and their marker bands derived from Raman images.
Figure 3: Schematic of the experimental procedure.
Figure 4: Variations in Raman spectra related to lignin content.
Figure 5: Raman images of Brachypodium distachyon.
Figure 6: Raman images of a poplar tension wood cross-section.
Figure 7: Raman images and spectra based on the same measurements shown in Figure 6.

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Acknowledgements

We thank P. Conchon and C. Coutand (INRA, Clermont-Ferrand, France) for providing the poplar cross-sections and M. Rüggeberg (ETH Zürich) for sharing the PEG-embedding protocol. The Max Planck Institute of Colloids and Interfaces (Potsdam, Germany) is acknowledged for providing the working environment to develop the Raman imaging approach on plant cell walls. We also acknowledge the Austrian Academy of Sciences (APART programme) for financial support.

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Authors and Affiliations

  1. Department of Material Sciences and Process Engineering, Institute of Wood Science and Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

    Notburga Gierlinger

  2. Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

    Notburga Gierlinger

  3. Institute of Polymer Science, Johannes Kepler University Linz, Linz, Austria

    Notburga Gierlinger & Tobias Keplinger

  4. Institut National de la Recherche Agronomique (INRA), UMR 1318, Institut Jean Pierre Bourgin, Versailles, France

    Michael Harrington

  5. AgroParisTech, Institut Jean Pierre Bourgin, RD10, Versailles, France

    Michael Harrington

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  1. Notburga Gierlinger
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  2. Tobias Keplinger
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  3. Michael Harrington
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Contributions

N.G. was the principal investigator, developed the Raman imaging approach for plant cell walls and wrote the manuscript. T.K. performed the polarization experiment on the ramie fiber and M.H. developed and described the protocol for cryosectioning of plant cell walls.

Corresponding author

Correspondence to Notburga Gierlinger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Schematics of the most important steps of the Polyethylenglycol (PEG) embedding. (PDF 991 kb)

Supplementary Figure 2

Schematic picture of the Confocal Raman microscope (PDF 301 kb)

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Gierlinger, N., Keplinger, T. & Harrington, M. Imaging of plant cell walls by confocal Raman microscopy. Nat Protoc 7, 1694–1708 (2012). https://doi.org/10.1038/nprot.2012.092

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