Name: combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall
Uploaded: 2017-06-10T14:00:00
Description: Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

私たちは財政的支援のために中国科学技術省（2016YDF0600803）に感謝します。

サンプル調製<ol><li>植物サンプル（ 例えば、ポプラステム）から小さな組織ブロック（約3mm×3mm×5mm）を切断する。 </li><li>組織を沸騰している脱イオン水に30分間浸します。直ちに室温（RT）で30分間脱イオン水に移す。組織が容器の底に沈み、組織の空気が除去され、組織が軟化したことを示すまで、このステップを繰り返します。 注：このステップの前に下に沈むサンプルに?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.">Chapter 2.</a> Ionic Liquids in the Biorefinery Concept: Challenges and Perspectives. , 38-64 (2016).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+tissue+specific+compositions+of+bioenergy+feedstocks+Through+hyperspectral+Raman+imaging.">Understanding tissue specific compositions of bioenergy feedstocks Through hyperspectral Raman imaging.</a> Bio. 108 (2), 286-295 (2009).</li><li>Tolstik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+prediction+of+HCC+tissues+by+Raman+imaging+with+identification+of+fatty+acids+as+potential+lipid+biomarkers.">Classification and prediction of HCC tissues by Raman imaging with identification of fatty acids as potential lipid biomarkers.</a> J. 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植物細胞壁は、細胞コーナー（CC）、二次壁（S1、S2、およびS3層を有するSW）および化合物中間薄層（CML、中間薄層および隣接する一次層）を含むいくつかの層に組織化された複合体である壁面）であり、試料調製中に平坦な表面を得ることが困難になる。したがって、木材より複雑な構造を有する植物サンプル、特に草は、しばしば細かい切片化を可能にするために凝固する必要がある。 PE...

Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3. 
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing. 
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Microtome</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:199px;">Microm HM430</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Raman microscope</td>
			<td>Horiba Jobin Yvon</td>
			<td>Xplora</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oven</td>
			<td>Shanghai ZHICHENG</td>
			<td>ZXFD-A5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall

プラントバイオマスへのラマンイメージングの適用は、水溶液について空間的および組成的情報を提供することができるので、増加している。分析には通常、大量の試料調製は必要ありません。ラベルを付けることなく構造的および化学的情報を得ることができる。しかしながら、各ラマン像は数千のスペクトルを含む。これは、特に類似の化学構造を有する構成要素について、隠された情報を抽出する際に困難を生じさせる。この研究では、この問題に対処するために多変量解析を導入しています。このプロトコルは、植物細胞壁内のリグニン、セルロースおよびヘミセルロースを含む主成分を視覚化するための一般的な方法を確立する。このプロトコールでは、試料調製、スペクトル取得、およびデータ処理のための手順が記載されている。これは、試料調製およびデータ分析における操作者の技能に大きく依存する。このアプローチを使用することにより、非専門家によるラマン調査を行い、HIを得ることができる植物細胞壁分析のための高品質データおよび有意な結果を提供する。

図1は、植物細胞壁のラマンイメージングのための典型的なマイクロラマンシステムの概要を示す。一例として、ポプラ（ Populus nigra L.）の元のラマンスペクトルは、有意なベースラインドリフトおよびスパイクを有する（ 図2a ）。ラマンイメージングデータセット（APRI）の自動前処理方法を実行した後、これら2つ...

Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

このプロトコルは、ラマンイメージングおよび多変量解析を用いて、植物細胞壁のリグニン、セルロースおよびヘミセルロースを視覚化するための一般的な方法を提示することを目的とする。

ラマンイメージングと多変量解析を組み合わせて、植物細胞壁のリグニン、セルロース、およびヘミセルロースを視覚化する

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

セルロース紙ディスクおよび裸眼比色イムノアッセイにおけるそれらのアプリケーションへの抗体の共有結合

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

マンナン ペースを用いた植物細胞壁多糖の構造解析

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

分離とシロイヌナズナ由来のミトコンドリアの呼吸測定

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2 の 1: タンパク質・ タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

ミリ秒時間スケールでのタンパク質複合体のダイナミクスを調べるため体外蛍光共鳴エネルギー移動の使用

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

C.エレガンス体壁筋肉における生体内カルシウムイメージング

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

茶由来の新たに確立された細胞懸濁液培養における6種の全身殺虫剤の代謝に関する研究(カメリアシネンシスL.葉

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

ネイティブ自己蛍光に基づくsFLIMによる植物切片における蛍光プローブとリグニン間の相互作用の測定

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

単細胞薬の取り込み、代謝、および効果を研究するための統合ラマン分光法と質量分析プラットフォーム

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

植物マイクロRNAの検出と定量のためのRNAブロット分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...