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

Мы благодарим Министерство науки и технологий Китая (2016YDF0600803) за финансовую поддержку.

1. Подготовка образца <ol><li> Вырежьте небольшой образец ткани (около 3 мм х 3 мм х 5 мм) из образца растения ( например, стебель тополя). </li><li> Погрузите ткань в кипящую деионизированную воду в течение 30 мин. Немедленно переносите его в деионизированную воду при комнатной температуре (RT) ...

<ol>
	<li>Gonzalo, G. D., 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=Bacterial+Enzymes+Involved+in+Lignin+Degradation.">Bacterial Enzymes Involved in Lignin Degradation.</a> J. Biotechnol. 236, 110-119 (2016).</li><li>Rosatella, A. A., Afonso, C. A. 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. Cancer. Res. Clin. Oncol. 141 (3), 407-418 (2015).</li><li>Schrader, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Infrared and Raman spectroscopy: methods and applications. , (2008).</li><li>Gierlinger, N., 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=Imaging+of+plant+cell+walls+by+confocal+Raman+microscopy.">Imaging of plant cell walls by confocal Raman microscopy.</a> Nat. Protoc. 7 (9), 1694-1708 (2012).</li><li>Luca, A. C. D., 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=Online+fluorescence+suppression+in+modulated+Raman+spectroscopy.">Online fluorescence suppression in modulated Raman spectroscopy.</a> Anal. Chem. 82 (2), 738-745 (2009).</li><li>Schl&#252;cker, S., 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=Raman+microspectroscopy:+a+comparison+of+point,+line,+and+wide-field+imaging+methodologies.">Raman microspectroscopy: a comparison of point, line, and wide-field imaging methodologies.</a> Anal. Chem. 75 (16), 4312-4318 (2003).</li><li>Cooper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemometric+analysis+of+Raman+spectroscopic+data+for+process+control+applications.">Chemometric analysis of Raman spectroscopic data for process control applications.</a> Chemometr. Intell. Lab. Syst. 46 (2), 231-247 (1999).</li><li>Cheng, H. J., Hsiau, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+granular+agglomeration+mechanism.">The study of granular agglomeration mechanism.</a> Powder Technol. 199 (3), 272-283 (2010).</li><li>Zhang, X., 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=Method+for+removing+spectral+contaminants+to+improve+analysis+of+Raman+imaging+data.">Method for removing spectral contaminants to improve analysis of Raman imaging data.</a> Sci. Rep. 6, 39891 (2016).</li><li>Shinzawa, H., 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=Multivariate+data+analysis+for+Raman+spectroscopic+imaging.">Multivariate data analysis for Raman spectroscopic imaging.</a> J. Raman Spectrosc. 40 (12), 1720-1725 (2009).</li><li>Lawton, W. H., Sylvestre, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+modeling+curve+resolution.">Self modeling curve resolution.</a> Technometrics. 13, 617-633 (1971).</li><li>Zhang, X., 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=Method+for+automatically+identifying+spectra+of+different+wood+cell+wall+layers+in+Raman+imaging+data+set.">Method for automatically identifying spectra of different wood cell wall layers in Raman imaging data set.</a> Anal. 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Растительная клеточная стенка представляет собой композит, который состоит из нескольких слоев, включая угол ячейки (CC), вторичную стенку (SW, с слоями S1, S2 и S3) и составную среднюю пластинку (CML, средняя ламелла плюс смежная первичная Стенки), что затрудняет получение плоской поверхности ?...

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

Применение изображений Raman для выращивания биомассы возрастает, поскольку оно может предлагать пространственную и композиционную информацию о водных растворах. Анализ обычно не требует обширной подготовки проб; Структурная и химическая информация может быть получена без маркировки. Однако каждый рамановский образ содержит тысячи спектров; Это создает трудности при извлечении скрытой информации, особенно для компонентов с аналогичными химическими структурами. В этой работе представлен многомерный анализ для решения этой проблемы. Протокол устанавливает общий метод визуализации основных компонентов, включая лигнин, целлюлозу и гемицеллюлозу в стенке растительной клетки. В этом протоколе описаны процедуры подготовки образцов, спектрального сбора и обработки данных. Он сильно зависит от навыков оператора при подготовке проб и анализе данных. Используя этот подход, рамановское расследование может быть выполнено неспециалистским пользователем для полученияH-качества и значимых результатов для анализа клеточной стенки растений.

На рисунке 1 представлен обзор типичной системы микрораманов для рамановской визуализации стенки клетки растения. В качестве примера, исходные спектры комбинационного рассеяния тополя ( Populus nigra L.) имеют значительные базовые дрейфы и всплески ( <str...

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

Этот протокол призван представить общий метод визуализации лигнина, целлюлозы и гемицеллюлозы в растительных клеточных стенках с использованием рамановского изображения и многомерного анализа.

Объединение рамановских изображений и многофакторного анализа для визуализации лигнина, целлюлозы и гемицеллюлозы в стенке клетки растения

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

Ковалентного связывания антител с целлюлозой дисков бумаги и их применение в видимую невооруженным глазом колориметрические иммуноанализа

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

Структурные характеристики Маннан полисахаридов клеточной стенки в растений с использованием ПАСЕ

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

Изоляция и дыхательных измерения митохондрий от Резуховидка Таля

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 в 1: один шаг очищение сродства для параллельного анализа протеин протеина и белка метаболит комплексов

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

Использование в Vitro флуоресценции резонанс передачи энергии для изучения динамики белковых комплексов в масштабе времени миллисекунду

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

В Vivo Кальций Imaging в C. elegans мышцы стены тела

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

Изучение метаболизма шести системных инсектицидов в недавно созданной культуре подвески клеток, полученных из чая(Камелия Sinensis L.) Листья

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

Измерение взаимодействия между флуоресцентными зондами и лигнином в секциях растений sFLIM на основе автофлюоресценции коренных народов

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

Интегрированная Рамана спектроскопия и массовая спектрометрия Платформа для изучения одноклеточного потребления наркотиков, метаболизма и эффектов

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

Анализ РНК-блотов для обнаружения и количественной оценки микроРНК растений

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 ...