我々は、実りのコラボレーションと有用な議論をアンドリューキャロル、ブライトChaibang、Purbashaサルカール（エネルギーバイオサイエンス研究所、バークレー校）、Bahramパービン（ローレンスバークレー国立研究所）とヴィンセントL.チェンマイ（ノースカロライナ州立大学）を感謝。この作品は、エネルギーバイオサイエンス研究所によってサポートされていました。分子ファウンドリーで仕事は契約番号DE - AC02 - 05CH1123下で、米国エネルギー省の科学局、基礎エネルギー科学局によってサポートされていました。

1。サンプル準備<ol><li>ミクロトームで、和の植物試料、例えばポプラの幹木材やシロイヌナズナの茎をマウントします。 </li><li>ネイティブ組織から薄片を（通常は厚さ20μm）カット。 </li><li>ガラスの顕微鏡スライド上に植物のセクションを転送します。 </li><li> D 2 O中の植物のセクションを浸し、D 2 Oの蒸発を防ぐために顕微鏡スライド上にシールされているガラスのカバースリップでカバー植物のセクションでは、現在のイメージングのための準備ですか、それは将来使用するために格納することができます。 </li></ol> 2。サンプルの測定<ol><li>顕微鏡対物および/またはカバーガラスにイマージョンオイルを適用します。 </li><li>顕微鏡対物が直面しているカバースリップで、顕微鏡の圧電走査ステージ上の顕微鏡スライドを置き、固定します。 </li><li>高開口数の液浸顕微鏡の対物レンズを用いてカバースリップ（100倍、NA = 1.40）を介してサンプルを表示し、関心のサンプル領域を探します。 </li><li>他のすべての実験室、顕微鏡の光源をオフにした後、位置分解顕微測定は、10から30 mWの典型的なパワー（と試料にCWレーザーからバンドパスフィルタ処理された単色緑色の光を（λ= 532nm）を焦点を当てて行われる）セットアップの概略については、図1を参照してください。自家蛍光は、長い波長のレーザー光で励起が賢明かもしれない、その場合に有用な測定値を、禁止する可能性があるいくつかのサンプルで発生する可能性があります。 </li><li>後方散乱ストークスシフトしたラマン光は顕微鏡対物レンズによって収集され、共焦点セットアップの空間フィルタとして機能するダイクロイックミラー、ピンホール、、とロングパスフィルタを通過し、回折格子のスリットに集光され光をスペクトルに分散し、ラマンスペクトルを与え、冷却CCDカメラによって検出される分光計、。ポプラ材のラマンスペクトルは1,600、1,700 cm -1の間のスペクトル領域での特徴的なリグニンのバンドで、図2に示します。 </li><li>化学イメージングと空間的なリグニンの分布の可視化のため、二次元スペクトルのマップは、ラスタが圧電スキャンステージとレーザーの焦点を介してサンプルをスキャンし、各サンプル位置のためにラマンスペクトルを記録することによって取得されます。三次元スペクトルマップはレーザーの焦点がz軸方向に沿って連続して踏まれたために2次元の地図を積み重ねることにより生成されることがあります。 </li></ol> 3。データ解析<ol><li>化学イメージングとリグニンの可視化のため、収集されたデータは、MATLAB（MathWorks社、バージョン7.7）を用いて分析される。データは2つの空間次元とスペクトル信号の三次元で構成されている三次元のハイパーキューブ、に配置されている。 </li><li>リグニンの分析のために、1550、1,700 cm -1の間のスペクトル領域（図2を参照）とみなされます。リグニンの空間分布は（図3を参照）ベースライン補正スペクトルの1550 1700 cm -1にから強度を統合することにより可視化される。ベースラインの補正に代わるものとして、二次微分スペクトルを計算し、二次微分のピークは、分析に使用することができます。 </li><li> 。特にconiferaldehydeとコニアルコールの部分に関してはリグニンの局在と化学は、、さらに（図2、文献15のイラストを参照1600、1,700 cm -1の間にある三つのバンドの嵌合のガウスピーク下の面積を評価することによって分析することができる- 17）。 </li><li>異なるスペクトルマップ間の強度の正規化を参照クラスタリングの分類を、kは、手段で得られる平均的なルーメンのスペクトル、の2,500 cm -1の外因性のOD伸縮バンドのピーク高さとして使用して実行されます。これは非常に重要であり、一つ異なる測定、組織、サンプルおよび種間のリグニンのシグナル強度を比較することができます。 </li></ol> 4。代表的な結果ポプラ（ ポプラangustifolia）の幹木材の代表的なラマンスペクトルを図2に示されています。特徴的なリグニンのバンドは1,600、1,700 cm -1の間のスペクトルの領域で発見されています。例として、ポプラ材の断面におけるリグニンの空間分布を図3に示す。可視画像と比較すると、形態学的に異なる細胞壁領域が原因で別のリグニンの信号強度に明確に区別さとなる。高リグニンの信号強度は、化合物の中間ラメラの細胞の隅（CC）と、やや少ない、（CML）で観察される。実体のない低い、まだ、リグニンの量は、繊維のS2壁層内で観察される。リグニンの信号強度の変動は、特に繊維の繊維から、CC、CMLおよびS2内である程度まで見られる。我々の測定における横方向の空間分解能は〜300 nmである。データの品質の間木質化を比較するためによく適していますサンプルとさらにリグニン化学15を解剖する。 <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="図1" /> 図1：楽器のセットアップの概略図BP：バンドパスフィルター、DM：ダイクロイックミラー、PH：ピンホール、LP：ロングパスフィルター。 <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="図2" /> 図2：D 2 Oに記録されたポプラ（ ポプラangustifolia）の幹木材の代表的なラマンスペクトル 。強調表示されたスペクトルの面積は（また、挿入図を参照）リグニンと特異的に起因する3つの山を有するスペクトル領域をマークします。 <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="図3" /> 図3：1550 1700 cm -1のからラマン信号強度を積分して得られる、：ポプラ材の断面のラマンリグニンの画像（下）（可視画像上）。

<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. 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利害の衝突は宣言されません。

リグノセルロース系材料は、階層と構造と組成の両方に関して不均一である。詳細な特性評価のために化学物質過敏症を持っている分析ツール、空間分解能、およびネイティブのコンテキストでこれらの材料についての洞察を与えることが望ましいです。記載された方法は、リグニンと天然状態の近くでサンプルを染色またはラベリングすることなくサブミクロンである空間分解能を持つリ?...

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<td>microscope slides</td>
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<td>pointed brush</td>
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label-free in situ imaging of lignification in plant cell walls

会議増大するエネルギーを安全に要求し、効率的に地球規模の課題です。そのため、費用対効果の高い、持続可能な解決策を見つけるように努めるバイオ燃料生産の研究は、局所および重要な課題となっています。リグノセルロース系バイオマスは、液体バイオ燃料〜1-6の変換のためのバイオマスの主な情報源になろうとしています。しかし、費用対効果的かつ効率的にこれらの植物細胞壁の材料の反抗劣化はバイオ燃料や化学物質4の生産で使用するための大きな障害となって提示。特に、リグニン、複雑で不規則なポリフェニルプロパノイドヘテロポリマーは、リグノセルロース系バイオマスの収穫後の脱構築に問題となる。バイオ燃料用のバイオマスの変換の例では、それは発酵7の単糖を生成することを目的とプロセスの糖化を抑制する。工業用の植物バイオマスの有効利用は、実際には植物の細胞壁がlignifiedされる範囲に大きく依存している。リグニンの除去はコストがかかり、制限因子8、リグニンは、したがって、細胞壁の変換を改善するために重要な植物の品種改良と遺伝子工学の対象となっている。 植物細胞壁の木質化の正確な急速な特性評価を可能にする分析ツールは、繁殖集団の多数を評価するためにますます重要になる。このようなリグニンなどのネイティブコンポーネントの分離のための抽出手順は、重要な化学的および構造的な変更が9月11日をもたらし、必然的に破壊的です。 現場の方法で分析化学は、このようにリグノセルロース系材料の組成および構造解析のための非常に貴重なツールです。ラマン顕微鏡は、レーザー光子のエネルギーのシフトは分子振動に関連しているレーザー、からそのように、単色光の非弾性またはラマン散乱に依存し、試料の固有のラベルフリーの分子&quot;フィンガープリント&quot;を提示する手法である。ラマン顕微鏡は、天然状態に近いの化学組成と分子構造についての洞察を与えて、最小限のサンプル調製で非破壊で比較的安価な測​​定値を手に入れることができます。共焦点ラマン顕微鏡による化学イメージングは、以前は木材の細胞壁12-14にセルロースとリグニンの空間分布の可視化のために使用されています。これらの以前の結果に基づいて、我々は最近、野生型およびリグニン欠損トランスジェニックポプラtrichocarpa（ブラックコットンウッド）の幹木材15に木質化を比較するためにこの方式を採用している。 1600、1,700 cm -1に 、リグニンの信号強度と局在との間のスペクトル領域でのリグニンのラマンバンドが16,17の分析は、 その場でマッピングした。我々のアプローチは、リグニン含有量の違い、ローカライズ、および化学組成を可視化。最近では、我々は18のサブμmである横方向の解像度を持つシロイヌナズナの細胞壁ポリマーのラマンイメージングを示した。ここでは、この方法は、染色や組織の標識せずに植物細胞壁中のリグニンの可視化と異なる組織、試料や種の木質化の比較を得た提示されます。

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

共焦点ラマン顕微鏡法に基づく方法は、ラベルフリー植物細胞壁中のリグニンの可視化と異なる組織、試料や種の木質化の比較に支払うよう提示される。

ラベルフリーその場で</em植物細胞壁の木化の>イメージング

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

12.3K ビュー.  University of California, Berkeley. 共焦点ラマン顕微鏡法に基づく方法は、ラベルフリー植物細胞壁中のリグニンの可視化と異なる組織、試料や種の木質化の比較に支払うよう提示される。

ビデオ: 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光子顕微鏡を用いたマウスの胸腺のその場イメージングで

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 ...

生物学

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.

植物細胞壁の包括的な組成分析（リグノセルロース系バイオマス）パートI：リグニン

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.

植物細胞壁の包括的な組成分析（リグノセルロース系バイオマス）パートII：炭水化物

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.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

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.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

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.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

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.

結晶セルロース蓄積中の高解像度の定量化<em&gt;シロイヌナズナ</em&gt;ルーツは、組織特異的な細胞壁の変更を監視します

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).

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

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.

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

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.

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

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

ラベルフリーその場でイメージング