Andrew Carroll, Parlak Chaibang, Purbasha Sarkar (Enerji Biosciences Enstitüsü, Berkeley), Behram Parvin (Lawrence Berkeley Ulusal Laboratuarı) ve Vincent L. Chiang (North Carolina State University), verimli işbirlikleri ve yararlı tartışmalar için teşekkür ederiz. Bu çalışma, Enerji Biosciences Enstitüsü tarafından desteklendi. Moleküler Döküm İş Sözleşme No DE-AC02-05CH1123 altında, ABD Enerji Bakanlığı Bilim Ofisi, Temel Enerji Bilimler Ofisi, tarafından desteklenen oldu.

1. Numune Hazırlama <ol><li> Mikrotom, sulu bitki örnek, örneğin, kavak kök ahşap veya Arabidopsis thaliana kök monte edin . </li><li> Doğal doku ince bölümler (genellikle 20 mikron kalınlığında) kesin. </li><li> Bitki bölüm cam bir mikroskop lamı üzerine aktarın. </li><li> D 2 O bitki bölümünde etmesini sağlayınız ve bir cam kapak kayma, D 2 O buharlaşmasını önlemek için mikroskop lamı üzerine mühürlü ile kapak Bitki bölümü görüntüleme için hazır ya da gelecekte kullanılmak üzere saklanabilir. </li></ol> 2. Numune Ölçümü <ol><li> Mikroskop objektif ve / veya kapak kayma daldırma yağ sürün. </li><li> Yerleştirin ve kapağı kayması ile mikroskop objektif bakan, mikroskop piezoelektrik tarama sahnede mikroskop lamı güvenli. </li><li> Yüksek sayısal açıklık immersiyon objektifte kullanarak kapak kayma (100x, NA = 1.40) ile örnek ve ilgi örnek alan bulmak. </li><li> Diğer tüm laboratuvar ve mikroskop ışık kaynakları kapattıktan sonra, pozisyon çözülmesi microspectroscopic ölçümler tipik bir 10 ila 30 mW güç (örnek üzerine bir cw-lazer bant geçiren filtreden monokromatik yeşil ışık (λ = 532 nm) odaklanarak yapılır kurulum şematik bir bkz. Şekil 1). Otofloresans durumunda daha uzun dalga boyunda lazer ışığı ile uyarma tavsiye olabilir, yararlı ölçümleri yasaklamak, bazı örnekler görülebilir. </li><li> Arka dağınık Stokes kaymıştır Raman ışık mikroskop objektif tarafından toplanan, konfokal kurulum mekansal bir filtre olarak hizmet veren bir dikroik ayna, bir iğne deliği, ve longpass filtre geçer ve bir ızgaranın yarık içine odaklanmış spektrometre, ışık Işıksal dağınık ve Raman spektrumu vererek, soğutmalı bir CCD kamera tarafından tespit. Kavak ağacından Raman spektrumu, 1.600 ve 1.700 cm -1 arasında spektral bölgede karakteristik Lignin bantlar, Şekil 2&#39;de gösterilmiştir . </li><li> Mekansal lignin dağılımı kimyasal görüntüleme ve görselleştirme için, iki boyutlu spektral bir harita, raster piezoelektrik tarama aşaması ile lazer odak noktası üzerinden örnek tarama ve Raman spektrumu her bir numune pozisyonu için bir kayıt ile kazanılır. Üç boyutlu spektral haritalar lazer odak z yönünde ardışık adım olduğu için iki boyutlu haritalar istifleme tarafından oluşturulan olabilir. </li></ol> 3. Veri Analizi <ol><li> Kimyasal görüntüleme ve lignin görselleştirme için, toplanan veriler MATLAB (MathWorks, sürüm 7.7) kullanılarak analiz edilmektedir. Veri iki mekansal boyutları ve spektral sinyaller için üçüncü bir boyut oluşan üç boyutlu hiperspektral bir küp olarak düzenlenmiştir. </li><li> Lignin analizi için, 1550 ve 1700 cm -1 arasındaki spektral bölgede (bkz. Şekil 2) olarak kabul edilir. Lignin mekansal dağılımı 1.550 ile 1.700 cm bazal düzeltilmiş spektrumları (bkz. Şekil 3) -1 yoğunluğu entegre tarafından görüntülenmiştir. Bazal düzeltme için bir alternatif olarak, ikinci türev spektrumları hesaplanmış ve analiz için kullanılan ikinci türev doruklarına olabilir. </li><li> Coniferaldehyde ve koniferil alkol moieties konusunda özellikle lignin yerelleştirme ve kimya, 1.600 ve 1.700 cm -1 arasında bulunan üç gruplarından monte Gauss doruklarına altında kalan alan (bkz. Şekil 2 ve refs 15 iç değerlendirerek analiz edilebilir 17). </li><li> Farklı spektral haritalar arasında Yoğunluk normalleşme k-means kümeleme sınıflandırılması ile elde edilen ortalama lümen spektrumları, yaklaşık 2,500 cm -1 ekstrensek OD streching yüksekliğine bir referans olarak kullanılarak yapılır. Bu kritik ve bir farklı ölçümler, doku, örnekler ve türler arasında lignin sinyal intensitesi karşılaştırmanızı sağlar. </li></ol> 4. Temsilcisi Sonuçlar Kavak (Populus angustifolia) kök ahşap bir temsilcisi Raman spektrumu Şekil 2&#39;de gösterilmiştir. Karakteristik lignin bantları, 1.600 ve 1.700 cm -1 arasında spektral bölgede bulunur . Bir örnek olarak, bir kavak ahşap kesit ligninin mekansal dağılımı Şekil 3&#39;te sunulmuştur. Görüntü ile karşılaştırıldığında, morfolojik olarak farklı hücre duvarı bölgelerde, farklı lignin sinyal yoğunluğu nedeniyle açıkça ayırt hale gelir. Yüksek lignin sinyal yoğunluğu biraz daha az bileşik orta lamel (KML), hücre köşeleri (CC) gözlenen ve. Lignin, Aşağı, yine asılsız değil miktarda lifler S2 duvar tabakası içinde görülmektedir. Lignin sinyal şiddeti değişkenlik özellikle fiber fiber içinde bir dereceye kadar, CC, KML ve S2 bulundu. Ölçümlerde lateral mekansal çözünürlükte ~ 300 nm. Veri kalitesi arasındaki lignification karşılaştırmak için kendisini iyi ödünçdaha fazla lignin kimya 15 incelemek örnekleri. <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="Şekil 1" /> Şekil 1: enstrümantal kurulum şematik BP: bandpass; DM: dikroik ayna, PH: iğne deliği, LP: longpass filtre. <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="Şekil 2" /> Şekil 2: D 2 O kaydedilen kavak (Populus angustifolia) kök ahşap bir temsilcisi Raman spektrumu. Vurgulanan spektral alanı (da inset görmek) üç doruklarına lignin özellikle atfedilebilir olan spektral bölgede işaretler. <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="Şekil 3" /> Şekil 3: 1.550 ile 1.700 cm -1 Raman sinyal yoğunluğu entegre ederek elde edilen bir kavak ahşap kesit Raman lignin (alt) (görünür görüntü üst).

<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. 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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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Lignocellulosic malzemeleri ve hiyerarşik yapısı ve kompozisyon bakımından heterojen. Kimyasal duyarlılık, uzaysal çözünürlük ve derinlemesine bir karakterizasyonu analitik araçlar için bu malzemelerin yerli bağlamda içgörüler arzu edilir verir. Açıklanan yönteme lignin ve doğal durumuna yakın boyama ya da örneklerin etiketleme alt mikron uzaysal çözünürlüğü ile lignocellulosic bitki biyokütle lignification karşılaştırılması görselleştirme tanıyor. Minimum numune hazırlama gerek...

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<td>immersion oil</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

Toplantı artan enerji güvenli bir şekilde taleplerini ve verimli bir şekilde acil bir küresel bir sorundur. Bu nedenle, maliyet-etkin ve sürdürülebilir bir çözüm bulmak istiyor biyoyakıt üretimi araştırma, güncel ve kritik bir görev haline gelmiştir. Lignocellulosic biyokütle biyokütle sıvı biyoyakıt 1-6 dönüşüm için birincil kaynak olmaya adaydır. Ancak, bu bitki hücre duvar malzemelerinin maliyet-etkin ve verimli bir bozulma dinlemezlik, biyoyakıt üretimi ve kimyasallar 4 bunların kullanımı için önemli bir engel sunar . Özellikle lignin, karmaşık ve düzensiz bir poli-phenylpropanoid heteropolymer, hasat sonrası Yapısızlaştırmanın lignocellulosic biyokütle sorunlu olur. Örneğin biyoyakıt biyokütle dönüşüm, basit şekerler fermantasyon 7 üretmeyi amaçlayan süreçlerinde saccharification engeller. Endüstriyel amaçlar için bitki biyokütle etkin kullanımı, bitki hücre duvarı Odunlaşmış olduğu ölçüde büyük ölçüde bağımlı aslında. Ligninin kaldırılması, masraflı ve kısıtlayıcı faktör 8 ve lignin bu nedenle hücre duvarı dönüşüm geliştirmek için önemli bir bitki ıslah ve genetik mühendisliği hedef haline gelmiştir . Bitki hücre duvarlarının lignification doğru hızlı karakterizasyonu izin analitik araçlar çok sayıda üreme popülasyonlarının değerlendirmek için giderek daha önemli hale gelir. Lignin gibi doğal bileşenlerin izolasyonu için önemli kimyasal ve yapısal değişiklikler 9-11 hakkında Ekstraktif prosedürleri getirerek, kaçınılmaz olarak yıkıcı olan. Yerinde yöntemleri Analitik kimyasal böylece lignocellulosic malzemelerin kompozisyon ve yapısal karakterizasyonu için paha biçilemez bir araç . Raman mikroskobu, bir lazer, lazer fotonların enerji vardiya moleküler titreşimler ile ilgili olduğu gibi, esnek olmayan ya da Raman saçılması monokromatik ışık dayanır ve örnek içsel bir etiket serbest moleküler &quot;parmak izi&quot; sunan bir tekniktir. . Raman mikroskobu yerli durumuna yakın bir kimyasal bileşim ve moleküler yapısı içgörüler vererek, en az numune hazırlama ile tahribatsız ve nispeten ucuz ölçümleri gelemez. Konfokal Raman mikroskobu ile kimyasal görüntüleme, daha önce, selüloz ve ahşap hücre duvarları 12-14 lignin mekansal dağılımının görüntülenmesi için kullanılır olmuştur. Önceki bu sonuçlara dayanarak, son zamanlarda, vahşi tip ve lignin eksikliği olan transgenik Populus trichocarpa (siyah kavak) kök ahşap 15 lignification karşılaştırmak için bu yöntemi kabul ettiler . 1.600 ve 1.700 cm -1, lignin sinyal şiddeti ve lokalizasyonu arasındaki spektral bölgede lignin Raman bantları 16,17 Analiz in situ eşleştirilmiş. Bizim yaklaşımımız, lignin içerik farkları, yerelleştirme ve kimyasal bileşimi görüntülendi. En son, 18 alt-mikron lateral çözünürlüğe sahip Arabidopsis thaliana hücre duvarı polimerlerin Raman görüntüleme gösterdi. Burada, bu yöntem, boyama ya da dokuların etiketleme olmadan bitki hücre duvarlarında lignin görselleştirme ve farklı dokuları, numune veya türlerin lignification karşılaştırılması karşılayabilme sunulmaktadır.

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

Etiket lignin bitki hücre duvarlarında görselleştirme ve farklı dokuları, numune veya türlerin lignification karşılaştırılması sağladığını konfokal Raman mikroskobu dayalı bir yöntem sunulmaktadır.

Etiket- In situ</emBitki Hücre Duvarlar Lignification> Görüntüleme

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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Biology

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

12.4K Görüntüleme.  University of California, Berkeley. Etiket lignin bitki hücre duvarlarında görselleştirme ve farklı dokuları, numune veya türlerin lignification karşılaştırılması sağladığını konfokal Raman mikroskobu dayalı bir yöntem sunulmaktadır.

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.

2-Foton Mikroskop kullanarak Fare Thymus in situ Görüntüleme

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 ...

Biyoloji

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.

Bitki Hücre Duvarları kapsamlı Kompozisyonel Analizi (Lignocellulosic biyokütle) Bölüm 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 ...

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.

Bitki Hücre Duvarları Kapsamlı Kompozisyonel Analiz (Lignocellulosic biyokütle) Bölüm II: Karbonhidratlar

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.

Farelerde, pankreatik beta-hücre Kütle yerinde Niceleme

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.

Living Görüntüleme Hücre Şekli Değiştir Drosophila Embriyolar

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.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

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.

Apoptotik hücresi Temizliği görüntülenmesi sırasında Canlı<em&gt; Drosophila</em&gt; Embriyo

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.

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

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).

Hibrid-Cut: Dirençli Bir Bitki Doku Örnekleri Bir Geliştirilmiş Kesit Yöntemi

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.

Helianthus Annuus'ta Büyük Ölçekli Termal Görüntüleme Taraması ile Bitki Transpirasyonunun Yeni Regülatörlerinin Belirlenmesi

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 ...

Etiket- In situ Görüntüleme

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