著者らは、資金援助、NIH / NIDCR R00DE018212（SPH）、NIH/NIDCR-R01DE022032（SPH）、NIH / NIDCR T32 DE07306（AJ、JDL）、NIH / NCRR S10RR026645、（SPH）と、予防と修復歯科学の部門を認め、口腔顔面科学、UCSF。また、著者は、Xradia大学院フェローシップ（AJ）、Xradia社、プレザントン、カリフォルニア州を認める。 博士、著者は、データの後処理との彼女の援助のために博士がキャサリンGrandfield、UCSFに感謝します。スティーブン·ワイナーとギリ者Naveh、科学、レホボト、イスラエルのワイツマン研究所、 中のその場での負荷装置に固有の彼らの洞察に満ちた議論のための博士ロンシャハー、エルサレムのヘブライ大学、イスラエル。著者はまた、マイクロXCT その場で装填装置を使用するためのUCSFの生体材料とバイオマイクロCTイメージング施設に感謝したいと思います。

動物のハウジングと安楽死：このデモで使用されるすべての動物は動物実験使用委員会（IACUC）と国立衛生研究所（NIH）のガイドラインに基づいて病原体を含まない条件下で飼育した。 標準ハードペレットラット飼料と水アドリブで動物を提供する。 IACUCによって承認されたUCSFの標準的なプロトコルに従って、二酸化炭素窒息、両側性開胸の二段階法を介して動物を安楽死させる。組織の劣化を避けるために、動物の犠牲の24時間以内の生体力学的試験を行う。 1。ラット下顎または上顎の製造および解剖<ol><li>筋突起と関節突起プロセス（ 図1）15を含む全体の下顎を維持しながら、ゆっくりと膜状の組織や筋肉組織の添付ファイルを切断することによって、ラットの下顎骨を削除します。 </li><li>車で別々のhemimandiblesefullyメスの刃と下顎恥骨の繊維組織を切断する。 注：これらは、物理的に2 番目の臼歯の生体力学的試験を妨害した場合、冠動脈および下顎頭プロセス、および下顎骨の枝（ 図1）を除去しなければならない。 </li><li>モルのロードを妨げないよう髄室を公開することなく切歯を切った。 </li></ol> 2。 その場での圧縮荷重用の試料作製（図2） <ol><li> その場でのローディング装置 （ 図2A）でそれをロードする前に、実験試料よりも有意に硬い材料を用いてスチールスタブに試料を固定する。 注意：ポリメチルメタクリレート（PMMA）があれば、歯科Explorerを使用して除去し、本試験と超過試料を固定するために使用された。 </li><li>両方でストレートエッジを使用して原子間力顕微鏡の金属試料ディスクと金利のパラレルのモル（S）の咬合面の位置を合わせます平面（ すなわち近心-遠心と頬舌下）。 </li><li>臼歯の周囲の鈍器とトラフを作成します。 注：この空間は、余分な液体が含まれており、 その場での負荷の間に組織の水和を維持するための「堀」として機能する必要があります。 </li><li>歯科用コンポジットを使用した同心円状（ 図2B）または偏心（ 図2C）ロードに構築するために、歯の表面を準備します。 15秒間咬合面上の35％リン酸ゲルで目的の歯の表面をエッチングする。 </li><li>脱イオン水で十分にエッチング液をすすぎ、空気/水シリンジまたは圧縮空気ボンベを使用して表面を乾燥させる。エクスプローラで、薄い層で、オープンカスプに接着剤の滴を広げた。歯科用硬化光で複合を治す。 注意：コンポジットを含むすべてのステップは、ランプからの直接光せずに実行する必要があります。このような条件は、望ましくない重合プロセスを加速し、クーロンう複合体の適切な配置を妨げるdは。部屋の照明が許容される。 </li><li>細かいメスまたはカミソリの刃で隣接する歯から余分な接着剤を除去します。 </li><li>表面の調製後面には、流動性歯科用コンポジットを配置して、歯科Explorerを使用して、関心のモル（S）の溝にそれを広げた。 </li><li> 30秒間、歯科用硬化光にコンポジットを公開します。 </li><li>関心のモル（S）と30秒間光硬化の咬合面から、歯科用レジンコンポジットを使用して約3〜4程度の咬合の蓄積を成形。 </li><li>ストレートエッジと高速ハンドピースを使用して、すべての試料間で整合性のある負荷スキームを有効にするには、平らな面に平行に、複合の蓄積の上を減らします。 注：生体力学テスト中に、他の標本は、50 mg / mlのペニシリンをトリスリン酸緩衝液（TBS）に格納され、ストレプトマイシン15すべきである。 </li></ol> 3。ローディング装置ドリフトと剛性、材料特性差別化機能、繊維状合同の現場読み込み中 <ol><li> 図2（b）に示すように均一なロード用のロードステージのアンビル上の複合蓄積した試料、およびテストを固定します。 </li><li> （ 図2Bおよび2C）、同心または偏心荷重をチェックするために、有限の負荷に試料をロードすることによって、その後、複合材料の表面に咬合紙を置きます。 </li><li>試料水和を確実にするために、検体を中心に、TBSに浸したキムワイプを配置します。標本の周りトラフを行い、撮像中に水和さの臓器を維持するためにTBSでそれを埋める。 </li><li> hemimandibleの固定化以下の変位速度で所望のピーク負荷にモルを圧縮するDebenソフトウェアに入力ピーク荷重と変位速度。 注：材料は荷重変換器の感度= 0.1（時間をかけて圧縮される典型的な読み出しは反動負荷を含める必要がありますN）。ロード時や変位時から、圧縮された材料のための荷重-変位曲線は16〜18を取得する必要があります。荷重サイクルから収集されたデータを用いて、関節の諸特性を決定することもできる。関節の剛性は、変位曲線19対 ​​荷重の負荷フェーズの直線部分（データの最後の約30％）の勾配を取ることによって計算されるべきである。 </li></ol> 4。リンタングステン酸（PTA）と軟組織染色、PDL、 注：X線減衰のコントラストを高めるために、PDLは、5％のPTA溶液20で染色されるべきである。 <ol><li>きれいな1.8ミリリットルガラスカープルに埋め戻し、PTA染色液と注射器に充填カープルを置く。 </li><li>関心対象の分子の周囲の歯周組織に構造上の損傷を防止するために隣接する歯のPDL空間内に溶液をゆっくりと（5分/カープル）を注入する。 （注）上記の手順をbすべき溶液の約5浴室carpules（9 ml）を注入し、周囲の組織に流すされるまで電子を繰り返す。準備された標本はまた、残りのPTA溶液（8時間）中で一晩浸漬することができる。 </li></ol> 5。推奨されるμ-XCTスキャン設定次のスキャン設定でM-XCTを実行します。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td>対物倍率</td><td> 4X、10X </td></tr><tr><td></td><td> 1800の画像</td></tr><tr><td> X線管電圧</td><td> 75のkVp（PTA染色したサンプルでは50のkVp） </td></tr><tr><td></td><td> 8 W </td></tr><tr><td>露光時間</td><td> 〜8月25日秒* </td></tr><tr><td></td><td> 〜4μmの（4倍対物レンズ）、〜2ミクロン（10×対物レンズ）** </td></tr></tbody></table> *露光時間は、幾何学的形状と、試料とX線管のvoの光学密度に基づいて変化し得る間電圧。 **実際のピクセル解像度が若干源、試料、検出器の構成に基づいて異なるであろう。

<ol>
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著者らは、開示することは何もありません。

このプロトコルを確立する最初のステップは、剛体を用いて装填フレームの剛性の評価に関連している。結果に基づいて、剛性が著しく低い剛性値を有する標本のさらなる試験のためのローディング装置の使用を可能に有意に高かった。第二段階は、剛体、異なる架橋密度のPDMS材料、及び繊維状ジョイントを用いて生成された搬入出曲線の2つの相を用いることにより、異なる剛性値を区別?...

いくつかの実験方法は、可動結合の繊維状関節の生体力学を調査するために使用され続けている。歯の器官に特異的な生体力学の方法は、歪みゲージ1-3、光弾性法4,5、モアレ干渉法6,7、電子スペックルパターン干渉8、およびデジタル画像相関（DIC）9-14の使用を含む。本研究では、革新的なアプローチは、繊維接合部の内部構造（石灰化した組織とそのインタフェース軟らかい区域からなる、このような靭帯などの組織とのインターフェース） インビボ条件に相当する負荷でを露出させるためにX線を用いた非侵襲的イメージングを含む。マイクロX線顕微鏡に結合されたその場での装填装置が使用される。ロード時および荷重 - 変位曲線は、新たに採取したラットヘミ下顎骨内の関心のモルロードされるように収集することができる。 M1）無負荷時にロードされ、2の場合）に同心と偏心してロードされたこの研究で提示アプローチのAINの目標は、3次元での条件を比較することにより、歯の骨の形態の効果を強調することである。カット標本の必要性を排除し、湿潤状態で、全体完全な臓器で実験を行うためには、3D応力状態の最大保存を可能にする。これは様々な荷重シナリオの下での複合体の動的プロセスを理解する上で、調査の新しい領域を開きます。 本研究では、スプラーグドーリーラットの無傷の線維関節内の試験PDLバイオメカニクスのための方法は、最適な生物工学モデル系として考え継手について詳細に説明する。彼らは臓器レベルのバイオメカニクスに関連する実験は、関節の三つの重要な特徴を強調するために、水和された条件の下で咀嚼負荷のシミュレーションが含まれます。 3点が含まれます：変位対1）反力を：歯の歯槽ソケット内の変位と荷重への反動応答、2）3次元（3D）空間構成と形態計測：歯槽ソケット歯の幾何学的関係、および原因の変化に読み出し1及び2において3）の変化すなわち 、同心から偏心荷重に積載軸。提案手法の3つの基本的な読み出しは、機能的需要の変化、および/または疾患に起因するいずれかの脊椎動物における関節の適応性を調査するために適用することができる。前述の読み出しの変化、異なる負荷率で特異的に変位に反動的負荷との間の相関関係、および反動ロード時および荷重 - 変位曲線を生じたが、関節の生体力学の全体的な変化を強調するために適用することができます。提案されたプロトコルの有効性は、三次元形態計測および関節の全体的な生体力学に機械試験読取値を結合することによって評価される。

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			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
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			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
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in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

この研究は、新規バイオメカニクス試験プロトコルを示しています。このプロトコルの利点は、このようにシミュレートされた生理的負荷および湿潤条件下で内部構造要素の可視化を可能にする高分解能X線顕微鏡に結合されたその場でのローディング装置の使用を含む。実験の試料は、無傷の骨 - 歯周靭帯（PDL）歯繊維状の関節が含まれます。変位対1）反力：歯の歯槽ソケット内での変位と荷重に対するその反動的応答、2）三次元（3D）空間配置の結果は、それらは、臓器レベル生体力学に適用することができるように、プロトコルの三つの重要な特徴を示すであろうおよび形態計測：幾何学的な肺胞のソケットと歯の関係に起因すなわち 、同心から偏心荷重に積載軸の変化への読み出し1及び2において3）の変更。提案されたプロトコルの有効性は、機械的なテを結合することによって評価される三次元形態計測と関節の全体的な生体力学に読み出しを刺す。また、この技術は、従来の繊維接合部の断層像を取得する実験条件、具体的に反動的負荷を平衡化する必要性を強調する。これは、提案されたプロトコルは、 エキソビボ条件下での試験片の試験に限定され、軟組織の機械的応答を可視化する造影剤の使用は、組織および器官レベルの生体力学に関する誤った結論を導くことができることに留意すべきである。

定荷重下でのローディング装置「バックラッシュ」、「プッシュ」、剛性、およびシステムのドリフトの推定 バックラッシュ：サイクルのロードとアンロード部の間に、真のアンロード標本が離れてトップジョー（ 図3）から引っ張るすなわちとして、開始前にモーター内で逆ギヤその間3秒のポーズが存在する。この期間は...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

本研究では、繊維性関節生体力学するマイクロX線コンピュータ断層撮影法と結合され、その場ローディング装置においての使用が議論される。実験の関節バイオメカニクスでの全体的な変化との識別可能な読み出しが含まれます：変位対1）反力を、 すなわち歯の歯槽ソケット内の変位と荷重への反動応答、2）3次元（3D）空間配置および形態計測、 すなわち幾何学歯槽ソケット歯の関係、および3）により装填軸の変化に読み出し1,2の変化、 すなわち、同心または偏心荷重。

その場圧縮負荷と骨歯根膜歯繊維状合同の相関的非侵襲的イメージングで

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.2K ビュー.  University of California San Francisco. 本研究では、繊維性関節生体力学するマイクロX線コンピュータ断層撮影法と結合され、その場ローディング装置においての使用が議論される。実験の関節バイオメカニクスでの全体的な変化との識別可能な読み出しが含まれます：変位対1）反力を、 すなわち歯の歯槽ソケット内の変位と荷重への反動応答、2）3次元（3D）空間配置お?...

ビデオ: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分解能光ファイバMicroendoscopy用その場で細胞イメージング

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

生物工学

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

繊維状タンパク質をストレッチし、測定する新しい重力分光器の使用を実証

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

ネイティブ、繊維状の長い間隔と分節ロング間隔コラーゲンのin vitro合成

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

組織工学のための二軸メカニカルローディングバイオリアクターの設計

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS対応の非侵襲メタボリック·プロファイリング

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

繊維状足場に微小球を離すエレクトロ成長因子

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

最適化されたセットアップおよびヒステリシス測定場の磁区イメージングのためのプロトコル

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

非侵襲的診断のプロテアーゼ活動In Vivo検出するナノセンサー

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

相関光電子顕微鏡 (クレム) 追跡のウイルス蛋白質のイメージング関連するクライオ固定化細胞の構造

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...