Damos las gracias a Andrew Carroll, Chaibang brillante, Purbasha Sarkar (Energy Biosciences Institute, Berkeley), Bahram Parvin (Lawrence Berkeley National Laboratory) y Vincent L. Chiang (North Carolina State University) para colaboraciones fructíferas y útiles debates. Este trabajo fue apoyado por el Instituto de Biociencias de la Energía. Trabajo en la fundición molecular fue apoyada por la Oficina de Ciencia de la Oficina de Ciencias Básicas de Energía, del Departamento de Energía de EE.UU. bajo el contrato No. DE-AC02-05CH1123.

1. Preparación de la muestra <ol><li> Montar la muestra vegetal hidratada, por ejemplo, la madera del tallo o el tronco de álamo de Arabidopsis thaliana, en el microtomo. </li><li> Los cortes de secciones delgadas (generalmente de 20 m de espesor) a partir del tejido de origen. </li><li> Transferencia de la sección de la planta sobre un portaobjetos de vidrio. </li><li> Remoje la sección de la planta en D 2 O y se cubre con un cubreobjetos de cristal, que se sella en el portaobjetos del microscopio para evitar la evaporación de D 2 O. La sección de la planta ya está listo para la imagen o se puede almacenar para uso futuro. </li></ol> 2. Ejemplo de medición <ol><li> Aplicar aceite de inmersión para el objetivo del microscopio y / o la hoja de la cubierta. </li><li> Colocar y fijar el portaobjetos en la etapa de exploración piezoeléctricas del microscopio, con la hoja de la cubierta hacia el objetivo del microscopio. </li><li> Ver la muestra a través de la hoja de la cubierta con una alta apertura numérica de inmersión objetivo de microscopio (100x, NA = 1,40) y localice el área de la muestra de interés. </li><li> Después de apagar todas las demás fuentes de laboratorio y microscopio de luz, resueltos posición microspectroscopic mediciones se llevan a cabo centrándose paso de banda, filtrado luz monocromática verde (λ = 532 nm) de un cw-láser en la muestra con una potencia típica de 10 a 30 mW ( véase la Figura 1 para un esquema de la configuración). Autofluorescencia puede ocurrir en algunas muestras, las cuales pueden prohibir medidas útiles, en la que la excitación caso ya la luz del láser de longitud de onda puede ser aconsejable. </li><li> El back-luz dispersa Stokes-Raman cambiado es recogida por el objetivo del microscopio, pasa a través de un espejo dicroico, un agujero de alfiler, que sirve como un filtro espacial en la configuración confocal, y un filtro de paso largo, y se centra en la ranura de una rejilla espectrómetro, donde se espectralmente la luz dispersada y detectada por una cámara CCD enfriado, dando un espectro Raman. El espectro Raman de madera de álamo se muestra en la Figura 2, con bandas de lignina característica en la región espectral entre 1.600 y 1.700 cm -1. </li><li> Para obtener imágenes de productos químicos y la visualización de la distribución espacial de la lignina, un mapa espectral de dos dimensiones es adquirida por trama de exploración de la muestra a través del foco de láser con la etapa de exploración piezoeléctricos y el registro de un espectro Raman para cada posición de la muestra. Mapas en tres dimensiones espectrales pueden ser generados por apilamiento de mapas bidimensionales que se intensificó el enfoque de láser consecutivamente a lo largo de la dirección z. </li></ol> 3. Análisis de Datos <ol><li> Para la industria química y la visualización de imágenes de lignina, los datos recogidos se analizaron utilizando MATLAB (The Mathworks, la versión 7.7). Los datos se organizan en un cubo hiperespectrales en tres dimensiones, que se compone de las dos dimensiones espaciales y una tercera dimensión para las señales espectrales. </li><li> Para el análisis de la lignina, la región espectral entre 1550 y 1700 cm -1 se considera (ver Figura 2). La distribución espacial de la lignina se visualiza mediante la integración de la intensidad de 1.550 a 1.700 cm -1 del espectro de referencia con corrección (ver Figura 3). Como alternativa a la corrección del nivel basal, la segunda derivada de los espectros se pueden calcular y los picos de la segunda derivada para el análisis. </li><li> Localización de la lignina y la química, especialmente con respecto a coniferaldehyde y fracciones coniferil alcohol, pueden ser analizados mediante la evaluación del área bajo los picos de Gauss equipado de las tres bandas encuentra entre 1.600 y 1.700 cm-1 (véase el recuadro de la Figura 2 y 15 Refs. - 17). </li><li> Normalización de la intensidad entre los diferentes mapas espectrales se realiza tomando como referencia la altura del pico de la banda extrínseca DO se extiende alrededor de 2.500 cm -1 en el espectro de luz promedio, que se obtienen por k-means clustering clasificación. Esto es fundamental y permite comparar las intensidades de la lignina de la señal entre las diferentes mediciones, los tejidos, muestras y de especies. </li></ol> 4. Resultados representante Un espectro Raman representante de madera de álamo madre (Populus angustifolia) se muestra en la Figura 2. Bandas características de lignina se encuentran en la región espectral entre 1.600 y 1.700 cm -1. A modo de ejemplo, la distribución espacial de la lignina en la madera de álamo una sección transversal se presenta en la Figura 3. En comparación con la imagen visible, las regiones de células morfológicamente distintos de la pared se distingue claramente debido a la intensidad de la señal diferente de lignina. De alta intensidad de la señal de lignina se observa en las esquinas de células (CC) y, algo menos, en la lámina media compuesta (LMC). Cantidades inferiores, sin embargo, insustancial, no de la lignina se observan en la capa de la pared de las fibras de S2. La variabilidad de la intensidad de la señal de la lignina se encuentra en cierto grado en CC, CML y S2, en especial de la fibra a fibra. La resolución espacial lateral en nuestras mediciones es de ~ 300 nm. La calidad de los datos se presta muy bien para comparar entre lignificaciónmuestras y para diseccionar la lignina química 15. <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="Figura 1" /> Figura 1: Un diagrama esquemático de la configuración del instrumento BP:. Paso de banda de filtro; DM: espejo dicroico, PH: agujero de alfiler; LP: paso largo del filtro. <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="Figura 2" /> Figura 2: Un representante de espectro Raman de madera de álamo madre (Populus angustifolia) registrado en D 2 O. El área resaltada espectral (véase también el recuadro) las marcas de la región espectral que tiene tres picos específicamente atribuibles a la lignina. <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="Figura 3" /> Figura 3: imagen Raman lignina (abajo) de una madera de álamo sección transversal (arriba: la imagen visible), que se obtiene mediante la integración de la intensidad de la señal 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. 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>

Materiales lignocelulósicos son jerárquicos y heterogéneos con respecto a la estructura y composición. Para una herramientas de caracterización en profundidad de análisis que tienen sensibilidad a los químicos, la resolución espacial, y que proporcionan información sobre estos materiales en el contexto nativos son deseables. El método descrito permite la visualización de la lignina y la comparación de lignificación de la biomasa vegetal lignocelulósica con una resolución espacial que es sub-micras, sin ma...

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<td>microscope slides</td>
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<td>D2O</td>
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<td>immersion oil</td>
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<td>tweezers</td>
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<td>pointed brush</td>
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<td>microtome</td>
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label-free in situ imaging of lignification in plant cell walls

Energía satisfacer las crecientes demandas de forma segura y eficiente es un desafío urgente global. Por lo tanto, la investigación sobre la producción de biocombustibles, que busca encontrar soluciones rentables y sostenibles se ha convertido en una tarea de actualidad y crítica. Biomasa lignocelulósica es un punto de convertirse en la principal fuente de biomasa para la conversión a biocombustibles líquidos 1.6. Sin embargo, la terquedad de estos materiales de la pared celular de los vegetales a la degradación económica y eficiente ofrece un gran impedimento para su uso en la producción de biocombustibles y productos químicos 4. En particular, la lignina, una compleja e irregular poli-fenilpropanoides heteropolímero, se vuelve problemática a la deconstrucción post-cosecha de biomasa lignocelulósica. Por ejemplo en la conversión de biomasa para biocombustibles, que inhibe la sacarificación en los procesos dirigidos a la producción de azúcares simples para la fermentación de 7. El uso eficaz de la biomasa vegetal con fines industriales es, de hecho, depende en gran medida del grado en que está lignificada de la pared celular de las plantas. La eliminación de la lignina es un factor de costo y la limitación de 8 y la lignina se ha convertido en un mejoramiento de las plantas y el objetivo clave de la ingeniería genética con el fin de mejorar la conversión de la pared celular. Herramientas analíticas que permitan la caracterización precisa de una rápida lignificación de las paredes celulares de plantas cada vez más importante para la evaluación de un gran número de poblaciones que se reproducen. Procedimientos de extracción para el aislamiento de los componentes nativos como la lignina son inevitablemente destructiva, dando lugar a productos químicos significativos y modificaciones estructurales 9.11. Química analítica en los métodos in situ son herramientas muy valiosas para la caracterización de la composición y estructura de materiales lignocelulósicos. Microscopía Raman es una técnica que se basa en la dispersión inelástica o Raman de luz monocromática, como la de un láser, que se relaciona con el cambio de energía de los fotones de láser para las vibraciones moleculares y presenta una intrínseca sin etiqueta molecular &quot;huella digital&quot; de la muestra . Microscopía Raman puede permitirse mediciones no destructivas y de bajo costo en comparación con una preparación mínima de la muestra, dando ideas sobre la composición química y la estructura molecular de una cerca de estado nativo. Imagen química por microscopía confocal de Raman ha sido utilizado anteriormente para la visualización de la distribución espacial de la celulosa y lignina en las paredes celulares de madera 12/14. Basándose en estos resultados antes, han adoptado recientemente este método para comparar la lignificación de tipo salvaje y lignina deficiente transgénicos Populus trichocarpa (negro álamo) madre de madera 15. El análisis de las bandas Raman lignina 16,17 en la región espectral entre 1.600 y 1.700 cm-1, la intensidad de la señal de la lignina y la localización fueron asignadas en el lugar. Nuestro enfoque visualizar las diferencias en el contenido de lignina, la localización y composición química. Más recientemente, hemos demostrado de imágenes Raman de los polímeros de la pared celular en Arabidopsis thaliana con una resolución lateral que es sub-micras 18. En este caso, este método se presenta ofreciendo la visualización de la lignina en las paredes celulares vegetales y comparación de lignificación en los diferentes tejidos, muestras o especies sin manchas o el etiquetado de los tejidos.

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

Un método basado en la microscopía confocal de Raman que se presenta ofrece sin etiquetas de visualización de la lignina en las paredes celulares vegetales y comparación de lignificación en los diferentes tejidos, muestras o especies.

Sin etiquetas In situ Imágenes de lignificación de las paredes celulares vegetales

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

Research

JoVE Journal

Biology

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

12.3K vistas.  University of California, Berkeley. Un método basado en la microscopía confocal de Raman que se presenta ofrece sin etiquetas de visualización de la lignina en las paredes celulares vegetales y comparación de lignificación en los diferentes tejidos, muestras o especies.

Video: 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.

En imágenes in situ del timo de ratón utilizando 2-Photon Microscopía

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 ...

Investigación

Biología

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.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte I: La 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.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte II: Los hidratos de carbono

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.

Celular imagen Cambiar forma de vida Drosophila Los embriones

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.

Perfiles de Polímeros Glycan la pared celular vegetal utilizando 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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

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).

Híbrido-Cut: un método de seccionamiento mejorada para muestras de tejido de la planta 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.

Identificación de nuevos reguladores de la transpiración de plantas mediante el cribado térmico de imágenes a gran escala en 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 ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imágenes Libres De Etiquetas De La Dinámica De Almacenamiento De Lípidos En Caenorhabditis elegans Usando Microscopía De Dispersión Raman Estimulada

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...