We thank Andrew Carroll, Bright Chaibang, Purbasha Sarkar (Energy Biosciences Institute, Berkeley), Bahram Parvin (Lawrence Berkeley National Laboratory) and Vincent L. Chiang (North Carolina State University) for fruitful collaborations and helpful discussions. This work was supported by the Energy Biosciences Institute. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH1123.

1. Sample Preparation
<ol>
	<li>Mount the hydrated plant sample, e.g. poplar stem wood or Arabidopsis thaliana stem, in the microtome.</li>
	<li>Cut thin sections (typically 20 &mu;m thick) from the native tissue.</li>
	<li>Transfer the plant section onto a glass microscope slide.</li>
	<li>Soak the plant section in D2O and cover with a glass cover slip, which is sealed onto the microscope slide to prevent evaporation of D2O. The plant section is now ready for imaging or it can be st...

<ol>
	<li>Herrera, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonkers+about+biofuels.">Bonkers about biofuels.</a> Nat Biotechnol. 24, 755-760 (2006).</li><li>Himmel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomass+recalcitrance:+Engineering+plants+and+enzymes+for+biofuels+production.">Biomass recalcitrance: Engineering plants and enzymes for biofuels production.</a> Science. 315, 804-807 (2007).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54, 559-568 (2008).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+metabolism+'Tear+down+this+wall.">Physiology and metabolism 'Tear down this wall.</a> Curr Opin Plant Biol. 11, 233-235 (2008).</li><li>Ragauskas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+forward+for+biofuels+and+biomaterials.">The path forward for biofuels and biomaterials.</a> Science. 311, 484-489 (2006).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofuels.">Biofuels.</a> Curr Biol. 17, R115-R119 (2007).</li><li>Ralph, J., Brunow, G., Boerjan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lignins in Encyclopedia of Life Sciences. , (2007).</li><li>Chiang, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+rags+to+riches.">From rags to riches.</a> Nat Biotechnol. 20, 557-558 (2002).</li><li>Atalla, R. H., Agarwal, U. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+microprobe+evidence+for+lignin+orientation+in+the+cell+walls+of+native+woody+tissue.">Raman microprobe evidence for lignin orientation in the cell walls of native woody tissue.</a> Science. 227, 636-638 (1985).</li><li>Atalla, R. H., Agarwal, U. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+Raman+spectra+from+plant+cell+walls.">Recording Raman spectra from plant cell walls.</a> J Raman Spectrosc. 17, 229-231 (1986).</li><li>Fukushima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+syringyl+to+guaiacyl+ratio+in+lignin+biosynthesis.">Regulation of syringyl to guaiacyl ratio in lignin biosynthesis.</a> J Plant Res. 114, 499-508 (2001).</li><li>Agarwal, U. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+imaging+to+investigate+ultrastructure+and+composition+of+plant+cell+walls:+distribution+of+lignin+and+cellulose+in+black+spruce+wood+(Picea+mariana).">Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana).</a> Planta. 224, 1141-1153 (2006).</li><li>Gierlinger, N., Schwanninger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+imaging+of+poplar+wood+cell+walls+by+confocal+Raman+microscopy.">Chemical imaging of poplar wood cell walls by confocal Raman microscopy.</a> Plant Physiol. 140, 1246-1254 (2006).</li><li>Gierlinger, N., Schwanninger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+Raman+microscopy+and+Raman+imaging+in+plant+research.">The potential of Raman microscopy and Raman imaging in plant research.</a> Spectrosc Int J. 21, 69-89 (2007).</li><li>Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+in+situ+imaging+of+lignification+in+the+cell+wall+of+low+lignin+transgenic+Populus+trichocarpa.">Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa.</a> Planta. 230, 589-597 (2009).</li><li>Agarwal, U. P., Argyropoulos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Raman+Spectroscopy+as+Applied+to+Lignocellulosic+Materials.">An Overview of Raman Spectroscopy as Applied to Lignocellulosic Materials.</a> Advances in Lignocellulosics Characterization. , 201-225 (1999).</li><li>Agarwal, U. P., Ralph, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+ethylenic+residues+in+wood+and+TMP+of+spruce+by+FT-Raman+spectroscopy.">Determination of ethylenic residues in wood and TMP of spruce by FT-Raman spectroscopy.</a> Holzforschung. 62, 667-675 (2008).</li><li>Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+imaging+of+cell+wall+polymers+in+Arabidopsis+thaliana.">Raman imaging of cell wall polymers in Arabidopsis thaliana.</a> Biochem Biophys Res Comm. 395, 521-523 (2010).</li></ol>

Lignocellulosic materials are hierarchical and heterogeneous with regard to both structure and composition. For an in-depth characterization analytical tools that have chemical sensitivity, spatial resolution, and that give insights into these materials in the native context are desirable. The described method affords the visualization of lignin and comparison of lignification of lignocellulosic plant biomass with spatial resolution that is sub-&mu;m without staining or labeling of the samples in a close to native state....

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>microscope slides</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cover slips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>D2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>nail polish</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>immersion oil</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>tweezers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>pointed brush</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microtome</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>confocal Raman microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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label-free in situ imaging of lignification in plant cell walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Watch this Scientific Journal Video about Label-free in situ Imaging of Lignification in Plant Cell Walls at JoVE.com

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

label-free-in-situ-imaging-of-lignification-in-plant-cell-walls

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Biology

12.3K Views.  University of California, Berkeley. Un metodo basato sulla microscopia confocale Raman è presentato che offre etichetta senza visualizzazione di lignina nelle pareti delle cellule vegetali e il confronto di lignificazione nei diversi tessuti, campioni o specie.