Agradecemos a Andrew Carroll, brilhante Chaibang, Purbasha Sarkar (Energy Biosciences Institute, Berkeley), Bahram Parvin (Lawrence Berkeley National Laboratory) e Vincent L. Chiang (North Carolina State University) para colaborações frutíferas e discussões úteis. Este trabalho foi financiado pelo Instituto de Biociências de Energia. Trabalho na Fundição Molecular foi apoiado pelo Escritório de Ciência, Escritório de ciências básicas da energia, do Departamento de Energia dos EUA no âmbito do contrato n º DE-AC02-05CH1123.

1. Preparação da Amostra <ol><li> Monte a amostra vegetal hidratado, por exemplo, madeira de álamo-tronco ou caule Arabidopsis thaliana, no micrótomo. </li><li> Cortar secções finas (normalmente 20 mm de espessura) do tecido original. </li><li> Transferência da seção de plantas para uma lâmina de vidro. </li><li> Mergulhe a seção planta em D 2 O e cobrir com uma lamínula de vidro, que é selado a lâmina de microscópio para evitar a evaporação de D 2 O. A seção de plantas já está pronto para imagens ou pode ser armazenado para uso futuro. </li></ol> 2. Medição da amostra <ol><li> Aplicar óleo de imersão para a objetiva do microscópio e / ou a lamínula. </li><li> Lugar e garantir a lâmina de microscópio no palco scan piezoelétrico do microscópio, com a lamínula de frente para a objetiva do microscópio. </li><li> Ver a amostra através do lamínula usando uma alta abertura numérica objetiva do microscópio de imersão (100x, NA = 1,40) e localizar a área da amostra de interesse. </li><li> Depois de desligar todas as outras fontes de luz e microscópio de laboratório, a posição-resolvido microspectroscopic medições são realizadas focando bandpass filtrada luz monocromática verde (λ = 532 nm) de uma cw-laser sobre a amostra com um poder típico de 10 a 30 mW ( veja a Figura 1 para um esquema do setup). Autofluorescência pode ocorrer em algumas amostras, que pode proibir medições úteis, em que a excitação caso com mais luz de comprimento de onda do laser pode ser aconselhável. </li><li> O back-luz difusa Stokes-shifted Raman é coletado pela objetiva do microscópio, passa por um espelho dicróico, uma pinhole, que serve como um filtro espacial na configuração confocal, e um filtro longpass, e está focada na fenda de uma grade espectrômetro, onde a luz é espectralmente dispersa e detectada por uma câmera CCD refrigerado, dando um espectro Raman. Um espectro Raman de madeira de álamo é mostrado na Figura 2, com bandas de lignina característica na região espectral entre 1600 e 1700 cm -1. </li><li> Para imagens de química e visualização da distribuição espacial de lignina, um mapa bidimensional espectral é adquirida pela digitalização raster a amostra através do foco do laser com a fase de digitalização e gravação piezoelétrico um espectro Raman para cada posição da amostra. Mapas tridimensionais espectrais podem ser geradas pelo empilhamento de mapas bidimensionais em que o foco do laser foi reforçada consecutivamente ao longo da direção z. </li></ol> 3. Análise de Dados <ol><li> Para a indústria química e visualização de imagens de lignina, os dados coletados são analisados ​​usando MATLAB (MathWorks, versão 7.7). Os dados são organizados em um cubo tridimensional hiperespectrais, que é composto pelas duas dimensões espaciais e uma terceira dimensão para os sinais espectrais. </li><li> Para a análise de lignina, a região espectral entre 1550 e 1700 cm -1 é considerado (ver Figura 2). A distribuição espacial da lignina é visualizado através da integração da intensidade de 1.550 a 1.700 cm -1 dos espectros de linha de base corrigida (ver Figura 3). Como uma alternativa para correção de linha de base, da derivada segunda espectros podem ser computados e os picos de segunda derivativos utilizados para análise. </li><li> Lignina localização e química, especialmente com relação a coniferaldehyde e metades de álcool coniferil, podem ser analisados ​​através da avaliação da área sob os picos equipado Gaussian das três bandas encontrados entre 1.600 e 1.700 cm -1 (ver inserção da Figura 2 e 15 Refs. - 17). </li><li> Normalização intensidade entre diferentes mapas espectral é realizada utilizando como referência a altura do pico da banda OD extrínsecos alongamento cerca de 2.500 cm -1 nos espectros da luz média, que são obtidos por meio de classificação k-clustering. Isto é crítico e permite comparar as intensidades de sinal de lignina entre as medidas diferentes, tecidos, amostras e espécies. </li></ol> 4. Resultados representante Um espectro Raman representativo de madeira de álamo haste (Populus angustifolia) é mostrado na Figura 2. Lignina bandas características são encontradas na região espectral entre 1600 e 1700 cm -1. Como exemplo, a distribuição espacial da lignina em uma madeira de álamo seção transversal é apresentada na Figura 3. Em comparação com a imagem visível, regiões morfologicamente distintas parede celular se claramente distinguíveis devido às diferentes intensidades de lignina sinal. Alto sinal de lignina é observado nos cantos das células (CC) e, um pouco menos, no meio composto lamelas (CML). Quantidades inferiores, ainda não insubstancial, de lignina são observados dentro da camada S2 da parede das fibras. Variabilidade de lignina intensidade do sinal é encontrado em certa medida, dentro de CC, CML e S2, especialmente da fibra a fibra. A resolução lateral espacial em nossas medições é de aproximadamente 300 nm. A qualidade dos dados presta-se bem para comparar entre lignificaçãoamostras e para continuar a dissecar lignina química 15. <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="Figura 1" /> Figura 1: Um esquema da configuração instrumental BP:. Bandpass filtro; DM: espelho dicróico, PH: pinhole; LP: longpass filtro. <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="Figura 2" /> Figura 2: Um espectro Raman representativo de madeira de álamo haste (Populus angustifolia) registrados em D 2 O. A área realçada espectral (ver também o inset) marca a região espectral tendo três picos especificamente atribuíveis a lignina. <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="Figura 3" /> Figura 3: Raman imagem lignina (inferior) de uma madeira de álamo seção transversal (de cima: imagem visível), obtida pela integração da intensidade do sinal Raman de 1.550 a 1.700 cm -1.

<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. 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Materiais lignocelulósicos são hierárquicas e heterogêneo em relação à estrutura e composição. Ferramentas para uma aprofundada caracterização analítica que têm sensibilidade química, resolução espacial, e que dão insights sobre estas matérias no contexto nativas são desejáveis. O método descrito permite a visualização de lignina e comparação de lignificação da biomassa vegetal lignocelulósica com resolução espacial que é sub-M, sem manchas ou rotulagem das amostras em uma situação próx...

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

Reunião crescentes demandas de energia de forma segura e eficiente é um desafio urgente global. Portanto, a investigação sobre a produção de biocombustíveis, que procura encontrar soluções cost-effective e sustentável tornou-se uma tarefa tópica e crítica. Biomassa lignocelulósica é posicionado para se tornar a principal fonte de biomassa para a conversão em biocombustíveis líquidos 1-6. No entanto, a recalcitrância desses materiais da parede celular de plantas para a degradação do custo-efetiva e eficiente apresenta um grande impedimento para a sua utilização na produção de biocombustíveis e produtos químicos 4. Em particular, a lignina, complexo e irregular heteropolímero poli-fenilpropanóides, torna-se problemática para a pós-colheita desconstrução da biomassa lignocelulósica. Por exemplo, na conversão de biomassa para os biocombustíveis, inibe sacarificação dos processos destinados a produção de açúcares simples para fermentação 7. O uso efetivo de biomassa vegetal para fins industriais é de fato em grande parte dependente da medida em que a parede celular vegetal é lignificado. A remoção de lignina é um fator limitante caro e 8 e lignina, portanto, tornar-se um melhoramento de plantas chave e alvo da engenharia genética, a fim de melhorar a conversão da parede celular. Ferramentas analíticas que permitem a caracterização exata rápida lignificação da parede celular das plantas cada vez mais importante para avaliar um grande número de populações reprodutoras. Procedimentos extrativistas para o isolamento de componentes nativos, como a lignina são inevitavelmente destrutivas, trazendo química significativa e modificações estruturais 9-11. Química analítica nos métodos in situ são, portanto, ferramentas inestimáveis ​​para a caracterização composicional e estrutural de materiais lignocelulósicos. Microscopia Raman é uma técnica que depende de espalhamento inelástico ou Raman de luz monocromática, como a de um laser, onde a mudança na energia dos fótons do laser está relacionado às vibrações moleculares e apresenta uma intrínseca label-free &quot;impressão digital&quot; molecular da amostra . Microscopia Raman pode pagar medições não-destrutivas e relativamente barato com a preparação da amostra mínima, dando insights sobre composição química e estrutura molecular em uma situação próxima do estado nativo. Química de imagens por microscopia confocal Raman tem sido utilizado anteriormente para a visualização da distribuição espacial de celulose e lignina nas paredes celulares de madeira 12-14. Com base nesses resultados anteriores, temos recentemente adoptado este método para comparar lignificação no tipo selvagem e lignina deficiente transgênicos trichocarpa Populus (preto cottonwood) haste de madeira 15. Analisando as bandas Raman 16,17 lignina na região espectral entre 1600 e 1700 cm -1, intensidade de sinal de lignina e localização foram mapeados in situ. Nossa abordagem visualizadas diferenças no conteúdo de lignina, localização e composição química. Mais recentemente, demonstramos imagem Raman de polímeros da parede celular em Arabidopsis thaliana com resolução lateral que é sub-M 18. Aqui, este método é apresentado proporcionando a visualização de lignina na parede celular das plantas e comparação de lignificação em diferentes tecidos, amostras ou espécie, sem manchas ou rotulagem dos tecidos.

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

Um método baseado em microscopia confocal Raman é apresentado que proporciona rótulo sem visualização de lignina na parede celular das plantas e comparação de lignificação em diferentes tecidos, amostras ou espécies.

Label-free In situ Imaging de lignificação na parede celular das plantas

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

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Research

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Biology

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

12.4K visualizações.  University of California, Berkeley. Um método baseado em microscopia confocal Raman é apresentado que proporciona rótulo sem visualização de lignina na parede celular das plantas e comparação de lignificação em diferentes tecidos, amostras ou espécies.

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

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Na imagem situ da Thymus Mouse Usando 2-Photon Microscopia

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

Biologia

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte I: A lignina

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Análise da composição completa de paredes celulares das plantas (biomassa lignocelulósica) Parte II: Os hidratos de carbono

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Quantificação em situ da Missa das células beta pancreáticas em camundongos

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imagem Alterar Forma Celular em Viver Drosophila Os embriões

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling glicana de Polímeros da planta da parede celular usando Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificação de novos reguladores de transpiração vegetal por triagem de imagens térmicas em larga escala em Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...