著者は、彼のアドバイスとエクセターのバイオイメージング施設の大学のためにエクセター大学の教授ニコラススミノフを感謝したいと思います。資金は、英国バイオテクノロジー生物科学研究会議（助成基準BB/E002682/1）によって提供されていました。

以下のプロトコルは、 シロイヌナズナの葉の浸潤性封入剤としてPFDは使用するためのシンプルな方法を説明しますが、我々は、このメソッドは画像空気が豊富な組織が ​​望まれる様々なアプリケーションで使用される場合があることを期待しています。 1。 PFDの取付け葉サンプル<ol><li>ポリジメチルシロキサンのガス透過性のガスケット（PDMS、ノースカロライナ観測ジェル）と顕微鏡のスライドを準備します。 PDMSは、粘弾性ポリマーであり、実験の要件に合わせたチャンバーを提供するように成形することができます。 </li><li>空気とPFDは平衡化させます。これは、空気バブリングによってまたは空気で満たされたボトルのPFDの小さな体積を振ることによって達成することができる。 </li><li>デカントは、開いているシャーレにPFDと5分間PFD上に全体実生または切除された葉をフロートさせます。組織は、ガラス固化体組織（ 図2（））を彷彿とさせる半透明になるはずです。葉は、PFD、依への曝露前より暗くまたは明るく表示されることがあります照明条件や使用する組織の年齢で丁。 </li><li>空気平衡PFDとPDMSチャンバーを記入し、慎重にPDMSチャンバーにインキュベーションペトリ皿から組織サンプルを転送する。実験的な要件に応じてカバースリップと画像でスライドを密封する。 </li><li>注：PFDもカバースリップ上に構築されたオープンなチャンバー内にまたは灌流チャンバ内のよく実行し、シリコングリスと互換性がある。それはそれらを溶解するとPFDは、テフロンのコンポーネントと互換性がありません。 </li></ol> 2。代表的な結果： PFD -インキュベートした葉サンプルの顕微鏡検査は、空域の大部分が浸水していることを示している。 図2（b）は PFDに懸濁した組換えGFPが殺到する空域を示しています。それは葉がPFDとインキュベーションにより浸水していることは明らかであるが、空気のその時々ポケットが残る場合があります。水は、これらの条件下で葉をフラッディングしません。サンプルのLSCMはでインキュベートし、マウントさ<html> IR、水またはPFDが可能なイメージング（ 図3）の深さで水と空気以上に優位に立つことPFDを示しています。 図3に示す例では、空気、水とPFDマウントされているシロイヌナズナが cytosolicallyローカライズされた金星、35Sプロモーターの制御下に恒常的に発現されたYFP 11の変形を表現する葉を示しています。 PFDと水を比較するときに我々は、イメージングの深さでおおよそ2倍の増加を見ることができます。それは、PFDを使用するときに見られる精密な改善がレンズと使用される組織の種類の開口数によって変化すること、しかし、注意する必要があります。我々は、PFD治療苗における細胞質流動と根毛の伸長、健康な植物の各指標を見てきました。さらに、我々 は FV / Fmは 、植物12の光合成機能の測定は、イメージングに使用されるタイムスケール上の許容限度内であること2を示している。 3394fig1.jpg&quot;/&gt; 図1。植物の葉の複雑さと光学的影響 A.の解剖学的特徴を示すダイヤグラム表示光学セットアップに関連したシロイヌナズナの葉。使用される略語はobjです。 =対物レンズ、IMM。 =液浸液、COV。 =カバースリップ、MNT。 =封入、カット。 =キューティクル、広告。 EP。 =向軸表皮、ST。 =気孔孔、SP。 =エアスペース、PALなど=海綿状葉肉、。 =柵状葉肉、VB =維管束、広告。 EP。 =向軸表皮。細胞壁は黒い線で示されます。 <img alt="図2。" src="/files/ftp_upload/3394/3394fig2.jpg" /> 図2。 PFDは、容易にシロイヌナズナの葉を貫通する （）空気、水の中でインキュベートまたは5分間PFDは葉とライカMZ16F顕微鏡（ライカマイクロシステムズ（英国）（株）、ミルトン - ケインズ、英国）とライカDCF3000FXデジタルカメラを用いて画像化。 Joveのロゴは、アセテートフィルムに印刷し、あちこちに照らされたライトボックスで下にはM。露光時間は89 msであり、すべての画像を収集し、同じように処理されました。スケールバーは2mmを表す。 （b）の組換え緑色蛍光タンパク質を精製運ぶPFD（GFP）は、 生体内で空域を区切ります。 GFPは、偽色の緑で、赤は葉肉細胞を区切るクロロフィル自家蛍光を、表示するために使用されます。 （ 図2（b）許可とリトルジョンらで利用可能な完全な技術的な詳細に再現。2010 2）。スケールバーは25 mを表す<img alt="図3。" src="/files/ftp_upload/3394/3394fig3.jpg" /> 図3。 PFDは、葉に共焦点イメージングを向上させる 無傷のシロイヌナズナで細胞質局在化金星蛍光（緑色）とクロロフィルの自家蛍光（赤色）を示すLSCMの画像は、空気、水またはPFDで結像される葉。励起波長は514 nmであると蛍光発光は、金星は604 nmの518 nmで記録し、少なくとも657 nmの葉緑体のための679 nmの。各パネルは、5μmの間隔で取得した11共焦点画像のZ -スタックから取り出した単焦点セクションです。これらは、補足的なムービーを生成するために使用されている1μmの間隔で取得した59個のイメージのフルZ -スタックから抽出した。深さの測定は、上皮表面に対して与えられる。同様に処理された代表的なイメージは、表示されます。そのような顕微鏡の設定などの実験の詳細は、リトルジョンらに見られるものと同じです。、2010 2。スケールバーは20μmを表す。

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

利害の衝突は宣言されません。

これは、空気充填または光学的に複雑な組織の顕微鏡を向上させるためにシンプルで使いやすい手法です。私たちは、テクニックはいくつかの強力な利点を持っていることが示されていると我々は、それが空気が豊富な組織に関連する生物学的疑問を解明するために使用されることを願っています。例えばそれは、植物の葉肉や肺内の病原体の攻撃の研究のための自然な選択になるでしょう。我々はまた、技術の限界を認識しています。我々は、長いタイムスケールの実験中に優れた屈折率のマッチング、顕微鏡の他のモードでの使用、及びパーフルオロカーボン封入補充に取り組んでいます。我々はまた、PFCガスの主な利点の一つは、すなわち生物学的に不活性なフリップサイドを持っていること、しかし、認識する。 PFCは容易にまたそれらが容易にそのようなホルモン、薬物、および他の小分子やイオンのような関心の化合物を送達するために使用することはできないことを意味する生物学的分子を溶解しない。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> Perfluorodecalin </td><td> F2ケミカルズ</td><td> N / </td><td>順番に電話。 </td></tr><tr><td>カロライナ観測ジェル</td><td>生物ブレード</td><td> 13から2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

生体試料に深く高解像度の画像を取得する問題が広く1を認めている。このような植物の葉の海綿状の葉肉のような空気で満たされた組織または肺の脊椎動物、さらに困難は細胞成分との間の、細胞と空域の間及び生体組織や光学系の他の部分との間の屈折率の複数の遷移から生じる。また、屈折率の不一致は、蛍光物質の励起および蛍光顕微鏡における信号の放出の減衰につながる。我々は光学的にレーザパワーでダメージが増加するに頼ることなく、深さで焦点顕微鏡（LSCM）サンプル画像をスキャンするレーザーを改善し、最小限の生理的影響2を有する浸潤のイメージング媒体としてここにパーフルオロカーボンのアプリケーション、perfluorodecalin（PFD）を、記述する。我々は、その結果として光学的に複雑であるシロイヌナズナの葉組織、とPFDの使用のためのプロトコルを記述する構造（ 図1）。 PFDは、この使用3に適するよう、属性の数を持っています。 PFDの屈折率（1.313）が水（1.333）と同等ですし、空気（1.000）よりも細胞質ゾル（約1.4）のそれに近いです。さらに、PFDは非蛍光、容易に入手可能であり、非毒性です。それは洪水を可能にする気孔貫通4、のための- PFDの低い表面張力（19ダインcm -1には ）水（72ダインcm -1）のそれより低く、また上限（30ダインcm -1の 25）以下です。潜在的に破壊的な真空または界面活性剤の応用せずにスポンジ状の葉肉の空域。最後にと決定的に、PFDは、CO 2とガス交換がこうしてサンプルについての生理学的影響を最小限に抑え、浸水組織で維持できるようにするO 2を 、溶解させるための偉大な能力を持っています。これらのプロパティは、部分的液体呼吸と肺のインフレを含む様々な用途で使用されているる5,6、手術7、人工血8、増殖培地9、及び植物10の氷の結晶形成の研究の酸素化。現在のところ、それは、ライブ共焦点イメージングのための水または水性緩衝液に組織をマウントするのが一般的です。私たちは、封入剤として、PFDの使用は、既存の慣行の改善を表し、イメージングのためのライブ全体の葉のサンプルの簡単な製造を可能にすることを検討してください。

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

我々は浸潤性封入剤としてperfluorodecalinの使用を説明しています。これは、撮像の深さを改善するための簡単​​な方法です。<em&gt;シロイヌナズナ</em最小限の生理的影響と&gt;葉組織。

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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Research

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Biology

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K ビュー.  The University of Exeter. 我々は浸潤性封入剤としてperfluorodecalinの使用を説明しています。これは、撮像の深さを改善するための簡単​​な方法です。シロイヌナズナ葉組織。

ビデオ: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

アン EX - OVO</emイメージングとマイクロサーアプリケーションに適して>ニワトリ胚培養システム

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

の細胞特異的な分析<em&gt;シロイヌナズナ</em&gt;蛍光活性化細胞選別を使用した葉

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

のための簡単​​なシート光顕微鏡を設定する<em&gt;トトで</em&gt;イメージングの<em&gt; C.虫</em&gt;開発

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

植物の葉からのアポプラスト液の抽出のための浸透·遠心分離技術の使用<em&gt;インゲンマメ</em&gt;例として

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

定量的イメージング技術を用いたヘテロ細胞集団の識別と特徴付け

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

高スループット シロイヌナズナ種子の殺菌のための標準化された方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

ベンサミアーナ タバコ葉蛋白質蛋白質の相互作用ダイナミクスの一過性の決定のための試金補完ルシフェラーゼ イメージング

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

規制エキソサイトーシスの記者として FITC デキストランをイメージング

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

イメージングのための簡単な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉