我々は、グラーツ、オーストリアのオーストリア科学アカデミーで博士フレッドHecusのXRSのKriechbaumと生物物理学やナノシステム研究の研究所に感謝したいと思います。 LLとXWは賞号NRC-38-08から950の下で、ネリ - C賞番号DE-FG07-07ID14889、米国原子力規制委員会の下に、米国エネルギー省によって部分的にサポートされていました。 SWAXS楽器も受賞番号DE-NE0000325の下、米国エネルギー省によって部分的にサポートされています。

1。サンプルの調製<ol><li>試料容器から試料の一部を削除するために針を使用します* </li><li>サンプルで（2.2ミリメートルの最大径）キャピラリーを埋めるために針を使用します。キャピラリーは下から2と3 cmの間埋めなければならない。 </li><li>その先端にワックスを溶融することによって毛細血管を閉じます。 </li><li>システムからネジを外し、真空試料ホルダーを。 </li><li>フューズドエンド（ワックスエンド）により毛細血管を取ると、試料ホルダーに非溶融の端を挿入します。 </li><li>代わりに、システムとネジに戻しホルダーを配置します。 </li></ol>液体試料が毛細管内に配置する必要があり、一方（粉末試料を含む）*固体試料を直接、（必要なキャピラリなし）サンプルホルダーに置くことができます。 SWAXSマシンの起動 2。ソースコールドスタートアップ·プロシージャ<ol><li>安全シャッターボタンを押してシャッターを閉じてください。 </li><li>主電源を切り替えるボタンに緑を押して、 &quot;ON&quot;。 </li><li>緊急 &quot;シャットオフ&quot;ボタンが延長された位置にあることを確認します。 </li><li>緑色のボタンを押して主電源を切り替えます。 </li><li>すべてのインターロックがokあることを示し、インターロックLEDライトが緑色に点灯していることを確認します。 </li><li>ロードするためにタッチスクリーンを待ちます。いったんロードしており、メニューの &quot;R6&quot;を押します。 </li><li> &quot;ON&quot;の位置にスタンバイキーを回してください。 </li><li> &quot;ON&quot;の位置にX線キーを回してください。赤のX線光が点灯します。 </li><li>スタンバイレベル（30 kVと0.4ミリアンペア）を読み取るためにタッチスクリーンを待ちます。 </li><li> &quot;OFF&quot;の位置にスタンバイ安全キーをオンにします。タッチスクリーンの下部にある &quot;スタートサイクル&quot;ボタンを押します。 </li><li>公称電力（50kV及び1 mA）を読むためにタッチスクリーンを待ちます。異なる電力レベルが望まれる場合には、タッチスクリーン上で &quot;R4&quot;を押して、必要な設定を入力して設定画面を表示します。 </li></ol> 3。チラー手順<ol><li>電源SWIを回し温度のコントロールパネルの &quot;ON&quot;の位置にTCH。制御光と、LED温度表示が点灯します。 </li><li>チラーの &quot;ON&quot;の位置に電源スイッチを切ります。 </li><li>温度表示が読み取るまで待つ &quot;OFF&quot;にします。これは、スタンバイモードです。 </li><li>を押して、約4秒または温度表示されるまで、Enterボタン（リターン記号）を保持するには、実際の温度を示しています。 </li><li>温度を変更するには、上矢印または下矢印を押します。それは実際の値に戻ります前に設定した値が約8秒間の温度表示に表示されます。 </li><li>所望の設定値が表示されたらENTERボタンを押します。これは、その値を格納します。 </li><li>実際の温度が所望の設定値に達するまで待ちます。 </li></ol> 注：エラー&quot;E 01&quot;温度ディスプレイ上に表示されます場合はいつでも、チラーのシャットダウン指示に従ってください、詰め替えバスユニットタンクFILに精製水を注いでリットル、その後再びチラーの起動手順を実行します。 4。チラーのシャットダウン<ol><li>約4秒間 &quot;Enter&quot;キーを押します。 </li><li> &quot;OFF&quot;の位置に電源スイッチを切ります。 </li></ol> 5。真空をオンにする<ol><li>真空ドアが所定の位置に固定されていることを確認。 </li><li> &quot;ON&quot;の位置に真空1と2のノブを回します。 </li><li> VAP5真空計が1.5ミリバール未満の真空レベルを読み取るまで待ちます。 </li></ol> 6。検出器システムのセットアップ<ol><li>ガス圧を設定するには、減圧弁の主要な圧力計は少なくとも10バールであることを確認します。 </li><li>メインバルブを開きます。 </li><li>圧力が使用圧力ゲージで8バールに達するまでゆっくり動作圧力バルブを開きます。 </li><li> 2主弁を開く</li><li>ガス流量を調整するために、ガスコントロールパネルの垂直位置にノブを回すことにより、メイン圧バルブを開きます。 </li><li>流量を調整ニードルバルブ付速度計が約8バーを読み込むまで。 </li><li> &quot;ON&quot;の位置に主電源スイッチをオンにします。 LEDライトが点灯するはずです。 </li><li>高電圧を調整するには、 &quot;ON&quot;の位置にスイッチを回すことにより高電圧をアクティブにします。 LEDライトが点灯するはずです。 </li><li>約3.5kVをゲージに到達するまで徐々に電圧制御ノブを回します。 4kVのを超えてはいけません。 </li></ol> 7。キャリブレーション<ol><li>上記の実験手順は、既知のピークのサンプルで実行されます。 </li><li> ASA 3.3は、5つのピークとの強度対チャネルのグラフを取得するために使用されます。 </li><li>メニュー→[オプション]→[入力チャネルと対応する強度に移動します。 </li><li>最初の &quot;TPF&quot;タブ、ASA3に移動します。 </li><li>フィルター変更、トップは1ミリメートルニッケル（ビームフィルタ）を残しました。 </li><li>位置32000（範囲28,000〜32000、28000の周りクリティカル領域）を設定→真空下5ミリバールであり、検出器がレアであることを確認していることを確認し、位置に移動1K-10Kの間で丁。 </li><li>秒当たり約52カウントのため10秒間実行します。 </li><li>エネルギーウィンドウを、（またはしないフィルタの場合を含め、サンプルのみでエネルギーウィンドウを測定）を変更します。 10秒のために最終的な実行を設定します。 </li><li>一次ビーム（例えば、チャネル233）の場所を見つける。 </li><li>他の実行では、2 mmのタングステン（W、ビームストッパー）に再びフィルタと 30,000からのスタートに変更します。 </li><li>サンプルの他のすべてのピークの位置を探します。 </li><li> X線小角散乱を校正するためにImageJのマクロ·ソフトウェアを使用して- （ 図1）強度対チャネルから強度対Q（またはd間隔）への遷移を行う。 </li><li> WAXSを校正するためにImageJのマクロ·ソフトウェアを使用して-強度対チャネルから強度対Q（またはd間隔）への移行を行う（ 図2）。 </li></ol> 8。ソフトウェアの手順<ol><li>ライブデータを収集するために、ソフトウェアのASA 3.3を使用してください：ライブデータ→TPFタブ→ファイルを保存します。 </li><li> EasySWAX用Sは、[デバイスの設定]タブをクリックします。 </li><li> &quot;a&quot;を、ラムダ、およびdの値と同様に、重心を入力します。 </li><li>ポイントコリメーションを選択します。 </li><li>球状​​粒子の種類を選択します（それが平らな形をした粒子のロッドであることが知られている限り）。 </li><li>ギニエ-Plotタブをクリックします。 IとQ 2のグラフが表示されます。 </li><li>ほぼ線形勾配の部分を囲むように縦線をドラッグします。 </li><li>画面の下部には、R値が表示されます。これに次の検証要件があります。 </li><li>検証値が1と2の間になるまで接近垂直線をドラッグし続けています。これは主観的なものです。得られたR値、または回転半径は、推定値である。 </li></ol> 9。ソースのシャットダウン手順<ol><li>安全を確認して、速いシャッターは閉じています。 </li><li> &quot;ON&quot;の位置にスタンバイキーを回してください。 </li><li>スタンバイ·レベルの読み取りにタッチスクリーンを待ちます。 </li><li>トンでプレスR5ouchscreen。 </li><li> 0 Aに電流を低減</li><li> 0 Aに電圧を下げる</li><li> &quot;OFF&quot;の位置にX線キーを回してください。 </li><li>赤オフボタンを押して、主電源を &quot;OFF&quot;に切り替えます。 </li></ol>

<ol>
	<li>Bernad&#243;, P., Blackledge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology:+Proteins+in+dynamic+equilibrium.">Structural biology: Proteins in dynamic equilibrium.</a> Nature. 468, 1046-1048 (2010).</li><li>Zhang, F., Skoda, M. W. A., Jacobs, R. M. J., Martin, R. A., Martin, C. M., Schreiber, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Interactions+Studied+by+SAXS:+Effect+of+Ionic+Strength+and+Protein+Concentration+for+BSA+in+Aqueous+Solutions.">Protein Interactions Studied by SAXS: Effect of Ionic Strength and Protein Concentration for BSA in Aqueous Solutions.</a> J. Phys. Chem. B. 111, 251-259 (2007).</li><li>Maranas, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+environment+on+local+dynamics+of+macromolecules.">The effect of environment on local dynamics of macromolecules.</a> Current Opinion in Colloid &amp; Interface Science. 12, 29-42 (2007).</li><li>Stribeck, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-Ray Scattering of Soft Matter. , (2007).</li><li>Mertens, H. D., Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+proteins+and+complexes+using+small-angle+X-ray+solution+scattering.">Structural characterization of proteins and complexes using small-angle X-ray solution scattering.</a> J. Struct. Biol. 172 (1), 128 (2010).</li><li>Svergun, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-angle+X-ray+and+neutron+scattering+as+a+tool+for+structural+systems+biology.">Small-angle X-ray and neutron scattering as a tool for structural systems biology.</a> Biol. Chem. 391 (7), 737 (2010).</li><li>Putnam, C. D., Hammel, M., Hura, G. L., Tainer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+solution+scattering+(SAXS)+combined+with+crystallography+and+computation:+defining+accurate+macromolecular+structures,+conformations+and+assemblies+in+solution.">X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution.</a> Quat. Rev. Biophys. 40, 191-285 (2007).</li><li>Bonini, M., Fratini, E., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAXS+study+of+chain-like+structures+formed+by+magnetic+nanoparticles.">SAXS study of chain-like structures formed by magnetic nanoparticles.</a> Materials Science &amp; Engineering C-Biomimetic and Supramolecular Systems. 27 (5-8), 1377-1381 (2007).</li><li>Falletta, E., Ridi, F., Fratini, E., Vannucci, C., Canton, P., Bianchi, S., Castelvetro, V., Baglioni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tri-block+copolymer+templated+synthesis+of+gold+nanostructures.">A tri-block copolymer templated synthesis of gold nanostructures.</a> Journal of Colloid and Interface Science. 357 (1), 88-94 (2011).</li><li>Glatter, O., Scherf, G., Schillen, K., Brown, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Poly(ethylene+oxide)+Poly(propylene+oxide)+Triblock+Copolymer+(EO(27)-PO39-EO(27))+in+Aqueous-Solution.">Characterization of a Poly(ethylene oxide) Poly(propylene oxide) Triblock Copolymer (EO(27)-PO39-EO(27)) in Aqueous-Solution.</a> Macromolecules. 27 (21), 6046-6054 (1994).</li><li>Mittelbach, R., Glatter, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+structure+analysis+of+small-angle+scattering+data+from+polydisperse+colloidal+particles.">Direct structure analysis of small-angle scattering data from polydisperse colloidal particles.</a> Journal of Applied Crystallography. 31, 600-608 (1998).</li></ol>

特別な利害関係は宣言されません。

SWAXSシステムの比較の手順は、実験的な分析から決定されるべき多くの変数を考慮します。解析から得られているパラメーターは、サンプルと実験のセットアップに応じて、異なる目的に使用することができます。 WAXSが原子とミクロスケールの構造（ 例えば分子 ​​格子、単位格子の次元対称性）に焦点を当てているのに対してSAXSは、ナノスケールの大きさと物体の形状に関する?...

<table border="1"><tbody><tr><td> 製品の名前 </td><td> 会社 </td></tr><tr><td>システム3小と広角X線散乱（SWAXS）カメラ</td><td> Hecus XRSとIBN、 グラーツ、オーストリア</td></tr><tr><td> GNOM </td><td> D. Svergun EMBL-ハンブルクATSAS 2.3パッケージ</td></tr></tbody></table>

small and wide angle x-ray scattering studies of biological macromolecules in solution

本論文では、高分子の小型広角X線散乱（SWAXS）分析は、実験によって実証されています。と広い角度（典型的には&gt; 5°） - SWAXSは、X線を弾性小さな角度（5°典型的には0.1）でのNM-範囲内不均一試料によって散乱されるテクニックです。この手法は、形状、大きさ、高分子、半順序材料、ポアサイズ、表面対体積比の特性距離の分布に関する情報を提供します。広角X線散乱（WAXSが）に基づいて0.33 nmと0.49 nmの間のサンプルのさらに小さいブラッグ間隔を解決できるのに対し、小角X線散乱（SAXS）は、1〜200nmの高分子の構造情報を供給することができます特定のシステムのセットアップおよび検出器。間隔は、ブラッグの法則から決定され、波長と入射角に依存している。 SWAXS実験では、材料は固体であってもよいまたは液体および任意の組み合わせで、同じまたは別の材料の固体、液体または気体のドメイン（いわゆる粒子）が含まれる場合があります。 SWAXSアプリケーションは、非常に広範であり、すべての種類のコロイドが含まれます：金属、複合材、セメント、油、ポリマー、プラスチック、タンパク質、食品、医薬品を。固体試料は、厚さが約5mmに制限されています。 ラボベースSWAXS機器の使用は、この論文に詳述されている。 SWAXSシステムで利用可能なソフトウェア（ 例えば 、D. Svergun EMBL-ハンブルクとEasySWAXSソフトウェアによってGNOM-ATSAS 2.3パッケージ）と、実験は、与えられたサンプルについて関心の特定のパラメータを決定するために実施することができます。生体高分子実験の一例としては、選ばれたとの多数の方法により製造することができる水性水性緩衝液中の2重量％のリゾチームの分析である。試料の調製は、サンプル·セクションの準備には、以下のガイドラインに従っています。 SWAXS実験を通して、リゾチームの重要な構造的パラメータ、 例えば回転半径、分析することができます。

旋回、粒径や形状の半径、溶液構造因子、特定の内表面と細孔径、格子型で、ディメンション、および電子密度1：SAXSとWAXSは完全に次のパラメータを介して試料の構造情報を提供することができます。 SAXSとWAXSは、タンパク質ダイナミクスの研究2に適用することができます。 SWAXS実験の構造情報は、実験的に検出されたスペクトルおよびシステムの計算...

Watch this Scientific Journal Video about Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution at JoVE.com

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution | Bioengineering | Video

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

小さいと広角X線散乱（SWAXS）プロシージャのデモンストレーションは、生体高分子の研究に尽力さとなっています。特定の角度の方法と準備の計装および手順を使用することにより、SWAXSからの実験データは、巨大分子の原子とナノスケールの特性を表示します。

溶液中の生体高分子の小型·広角X線散乱による研究

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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17.9K ビュー.  Rensselaer Polytechnic Institute. 小さいと広角X線散乱（SWAXS）プロシージャのデモンストレーションは、生体高分子の研究に尽力さとなっています。特定の角度の方法と準備の計装および手順を使用することにより、SWAXSからの実験データは、巨大分子の原子とナノスケールの特性を表示します。

ビデオ: Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

核酸、タンパク質と低分子のReagentless検出用電気化学DNAバイオセンサーの作製

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

生物工学

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

その時 液体中の生物学的集合体のTEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

多層魚の鱗の特性（<em&gt; Atractosteusヘラ</em&gt;）ナノインデンテーション、X線CT、FTIR、およびSEMを用いて

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

小角中性子回折計KWS-2でのナノおよびマイクロメータサイズからワイド長さスケール上でソフトマターと生物学的システムを学びます

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

血管新生および血管組織工学を研究するための小さな動物モデルにおける動静脈（AV）ループ

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

生物学的設定における生成と観測化学発光のための新しい技術

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

非常に小さいスペースで先端成長植物細胞の伸長機能を勉強するマイクロ流体デバイスの開発

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Nanoplasmon の散乱と低倍率の顕微鏡イメージングによる腫瘍由来エキソソームの定量化

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

ヒト線維筋組織を可視化するホスホトン酸調製のマイクロX線コンピュータ断層撮影の使用

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...