R21AG042066：この研究は、NIHからの助成金によってサポートされていました。私たちは、画面キャプチャのビデオを作成する際の彼の貢献のためにフィリップ·シュルツに感謝したいと思います。

<ol>
	<li>Hedieh, B., Sharareh, E., Philip, S., Michael, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+recombinant+antibodies+against+specific+protein+morphologies+using+atomic+force+microscopy+and+phage+display+technologies.">Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.</a> Protein Engineering Design and Selection. 19, 497-502 (2006).</li><li>Emadi, S., Barkhordarian, H., Wang, M. S., Schulz, P., Sierks, M. 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V., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-oligomeric+A&#946;+Single-chain+Variable+Domain+Antibody+Blocks+A&#946;-induced+Toxicity+Against+Human+Neuroblastoma+Cells.">Anti-oligomeric A&#946; Single-chain Variable Domain Antibody Blocks A&#946;-induced Toxicity Against Human Neuroblastoma Cells.</a> Journal of Molecular Biology. 384, 917-928 (2008).</li><li>Boddapati, S., Levites, Y., Sierks, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+&#946;-Secretase+Activity+in+Alzheimer's+Disease+Cell+Models+with+Single-Chain+Antibodies+Specifically+Targeting+APP.">Inhibiting &#946;-Secretase Activity in Alzheimer's Disease Cell Models with Single-Chain Antibodies Specifically Targeting APP.</a> Journal of Molecular Biology. 405, 436-447 (2011).</li><li>Boddapati, S., Levites, Y., Suryadi, V., Kasturirangan, S., Sierks, M. 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The authors have nothing to disclose.

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer&#8217;s, Parkinson&#8217;s, ALS and FTD1,2,3,4,5,6,7,8,9,10,11. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress ...

タンパク質変異体の存在は、アルツハイマー病、パーキンソン病、ALSおよび前頭側頭型認知症（FTD）1,2,3,4,5,6,7,8,9などの神経変性疾患を含む多くの疾患の進行の要因として関与している、10,11。タンパク質βアミロイドおよびα-シヌクレインのオリゴマー形態は、それぞれ2,3,4,5、アルツハイマー病及びパーキンソン病の原因で有毒種であると考えられている。 TAR DNA結合タンパク質43（TDP-43）の凝集体は、ALSとFTD 12,13,14にリンクされている。したがってそのような選択的に異なるタンパク質変異体は、診断マーカーおよび潜在的な治療薬として機能するための強力なツールであることができるターゲットにすることができる抗体などの試薬。本研究では、しかし、本論文で概説技術はprotの広い範囲に対して、試薬の分離に適用可能であるべきである、選択的にALSに関与TDP-43タンパク質の変異体を結合した​​試薬の開発に注力EIN変種。 TDP-43の細胞質の凝集は、ALS 15,16,17,18,19における病理学的特徴として同定されている。これは、サイトゾルおよび核15,17間を移動する傾向があるが、典型的にTDP-43は、正常な個体からのすべての細胞の核内に見出される。しかし、ALSにTDP-43の形態は、疾患の進行16,20の間に核から細胞質へのTDP-43の動きを示唆して核内に見つかったより低い濃度で選択したニューロンとグリアの細胞質で検出された集約。 TDP-43の凝集がALS症例の大多数において見出されているが、1％-2合計ALS症例の％（または家族性ALS症例の15％-20％）の変異にリンクされているので、すべての場合を考慮していないスーパーオキシドジスムターゼ1（SOD1）遺伝子15,17。そのためALS症例の大多数においてTDP-43の重要な役割のために、ここでは選択的にTDP-43変異体にも結合することができる抗体ベースの試薬の開発に注力私たちの小説AFMベースのバイオパニング技術を利用して、人間のALSの脳組織に存在する。 当初我々は、抗体結合ドメインの多様なレパートリーを必要としています。我々は、3つの異なるファージディスプレイ単鎖可変ドメイン抗体フラグメント（scFvの）ライブラリ（トムリンソンIとJとシートライブラリ21）を合わせた。パニングプロセスは、負および正のパニング段階に分けられる。ライブラリーからのファージを、最初に、複数のオフターゲット抗原が排除されるに反応するファージその間ネガティブパニングプロセスに供される。各オフターゲット抗原に対するネガティブパニングの各ラウンドが終了した後、プロセスは、オフターゲット抗原に結合する全てのファージが除去されたことを確認するためにAFMイメージングによってモニターされる。のみすべての反応性のファージを除去したAFMイメージングによって確認した後、我々は次の目標に進んでください。 ALSに関与TDP-43の変種に対する試薬を分離するために、我々は以下の負のパンニングの抗原を利用：<html>タンパク質に弱く又は非特異的に結合するファージを除去するための1）BSA。凝集したタンパク質のジェネリック構造要素に結合するファージを除去するための2）凝集したα-シヌクレイン。 3）ヒト脳組織ホモジネートは、任意のタンパク質または健康なヒトの脳組織の死後のサンプル中に存在する他の成分に結合するファージを除去する。 4）健康なヒトの脳に関連するすべてのTDP-43のフォームに結合するファージを除去するために健康なヒトの脳からTDP-43を免疫沈降した。 5）非ALSの病態に関連TDP-43変異体を結合するファージを除去するためにFTD脳ホモジネートからTDP-43の単離された免疫沈降した。すべてのオフターゲット抗原に対する反応性のすべてのファージを除去した後、我々は、この場合には、TDP-43は、ヒトのALS脳組織から免疫沈降し、目的の抗原に結合する抗体断片が単離され、その間に正のパニング段階に進む。これらの単離された抗体は、TDP-43の集約または改変された形態への反応性であり得る。 &quot;&gt;従来のファージパニングは正のパニング段階22,23に主に焦点を当てている。通常、目的の標的が固定化され、ファージライブラリーを添加し、結合したファージは、この増幅は、インキュベーション工程をファージを次に増幅される。溶出し、再びターゲットに追加このプロセスのバリエーションは、標的抗原の広い範囲に対する抗体試薬を単離するために広く使用されてきたが、通常は正の結合ファージの割合を増加させるために数回繰り返され、それらは一般的に精製された標的抗原24,25,26を大量に必要とし、本プロセスは、ターゲット抗原の微量必要一方、 図27は 、ここで説明するプロトコルは、精製およびパンを必要とせずに非常に低い濃度で存在している選択的に結合する標的抗原は、に対して直接行うことができる試薬を単離することができる複雑な組織サンプル中に存在する抗原。確認されるように徹底的なネガティブパニングプロトコルの使用AFMにより正の抗原に対する単離されたクローンを選択的に精製または濃縮なくても標的に結合する必要があることを保証します。 Kasturiranganら（2003）は、ターゲット5ナノグラム濃度を用いてオリゴマーβ-アミロイドに対して反応性の抗体を単離するために類似の負及び正のバイオパニングプロセスを行った。ここでは、選択的に結合する疾患特異的タンパク質がヒト組織サンプルから直接改変試薬の生成を有効にするには、このプロセスに展開します。今後の研究では、我々はさらにだけでなく、ここで分離された試薬の診断価値を調査するだけでなく、ALSの治療のためにそれらの治療の関連性を評価していきます。 全体として、本発明の新規なAFMベースのバイオパニング技術は、タンパク質精製または修飾を必要とせずに、任意の生物学的材料の任意の疾患特異的タンパク質変異体の単離に適用可能であるべきである場合でも、標的抗原concentratioナノは極めて低い。

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent/ Equipment</td>
			<td style="width:287px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:212px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:323px;">Tomlinson I and J Libraries</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sheets Library</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2xYT</td>
			<td style="width:287px;">BD Sciences</td>
			<td style="width:119px;">244020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glucose</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0188-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ampicillin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0339-25G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">KM13 Helper Phage</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Kanamycin</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">5880</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Polyethylene Glycol 8000</td>
			<td style="width:287px;">OmniPur</td>
			<td style="width:119px;">6510</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Chloride</td>
			<td style="width:287px;">Macron</td>
			<td style="width:119px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium Phosphate Dibasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0404-1KG</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Chloride</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">PX1405-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Potassium Phosphate Monobasic</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0781-500G</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TG1 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria-Bertani Agar</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;">1.10283.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bovine Serum Albumin</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0332-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">STEN buffer</td>
			<td style="width:287px;">Crystalgen Inc.</td>
			<td style="width:119px;">33429775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Immunotubes</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">470319</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Mica</td>
			<td style="width:287px;">Spruce Pine Mica</td>
			<td style="width:119px;">24365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tween 20</td>
			<td style="width:287px;">EMD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Trypsin</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0303</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethylamine</td>
			<td style="width:287px;">Sigma</td>
			<td style="width:119px;">T-0886</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Glycerol</td>
			<td style="width:287px;">Amresco</td>
			<td style="width:119px;">0854-1L</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNA Plasmid Prep Kit</td>
			<td style="width:287px;">qiagen</td>
			<td style="width:119px;">27106</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Non-Fat Milk Powder</td>
			<td style="width:287px;">Carnation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96-Well High Binding ELISA Plate</td>
			<td style="width:287px;">Costar</td>
			<td style="width:119px;">3590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-M13 HRP</td>
			<td style="width:287px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">27-9421-01</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:323px;">ELISA Femto Chemiluminescence Substrate Kit</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">37074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-TDP 43 Polyclonal Antibody</td>
			<td style="width:287px;">ProteinTech</td>
			<td style="width:119px;">10782-2-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">A/G Agarose Beads</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HB 2151 Cells</td>
			<td style="width:287px;">MRC (Cambridge, England)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropylthiogalactoside</td>
			<td style="width:287px;">Teknova</td>
			<td style="width:119px;">13325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">9e10 HRP</td>
			<td style="width:287px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrocellulose Membrane</td>
			<td style="width:287px;">Biorad</td>
			<td style="width:119px;">162-0115</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Centrifuge</td>
			<td style="width:287px;">Thermo Scientific</td>
			<td style="width:119px;">Sorvall RC 6+</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoscope IIIa Atomic Force Microscope</td>
			<td style="width:287px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AFM Probes</td>
			<td style="width:287px;">VistaProbes</td>
			<td style="width:119px;">T300R-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel atomic force microscopy based biopanning for isolation of morphology specific reagents against tdp-43 variants in amyotrophic lateral sclerosis

タンパク質変異体は、筋萎縮性側索硬化症（ALS）、パーキンソン病におけるα-シヌクレインおよびアルツハイマー病におけるβアミロイドおよびタウにおけるTDP-43を含む多くの疾患において重要な役割を果たしているので、選択的に標的とすることができる形態学的試薬を開発することが非常に重要であるこれらの疾患特異的タンパク質が疾患の病状および潜在的な診断および治療用途のためにこれらの変異体の役割を研究する変異体である。我々は、選択的に、疾患特異的タンパク質の変異体を認識する試薬の単離を可能にする新規の原子間力顕微鏡（AFM）ベースのバイオパニング技術を開発した。プロセスに関与する2つの主要な相、負および正のパニング段階がある。負のパン·フェーズでは、オフターゲット抗原に反応性のあるファージは、厳選されたオフターゲット抗原の一連を利用し、サブトラクティブパニングの複数ラウンドを排除している。負の重要な機能パニング段階プロセスを監視し、すべての望ましくないファージ粒子が除去されていることを確認するために、AFMイメージングを利用している。正のパニング段階のために、関心対象の標的抗原は、雲母表面上に固定され、結合したファージを溶出させ、選択的に標的抗原に結合するファージを同定するためにスクリーニングされる。標的タンパク質の変異体は、適切な負のパンコントロールが使用されている提供精製する必要はない。複雑な生物学的物質に非常に低い濃度でのみ存在するタンパク質変異体は、正のパニング段階で利用することができるターゲット。この技術を適用することにより、我々は選択的に、人間のALSの脳組織に見られるTDP-43のタンパク質変異体に対する抗体を獲得した。我々は、このプロトコルは、選択的に異なる生物学的プロセスおよび疾患の多種多様に存在するタンパク質の変異体に結合する試薬を生成することに適用可能であるべきであることを期待する。

図1では、回路図は、我々はイムノを使用して私たちのライブラリからオフターゲット抗原に結合するファージを削除したことにより、負のパニングプロセスを示しています。これは一般的なブロッキング剤であり、このターゲットと非特異的に反応するすべてのファージは将来のイムノアッセイにおいて問題となるので、私たちは、最初にBSAで始めた。次に、凝集したタンパク...

Watch this Scientific Journal Video about Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis at JoVE.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

筋萎縮性側索硬化症におけるTDP-43の変異体に対する形態学的試薬の分離のための新規原子間力顕微鏡ベースのバイオパニング

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

novel-atomic-force-microscopy-based-biopanning-for-isolation

Research

JoVE Journal

Bioengineering

8.7K ビュー.  Arizona State University. Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

ビデオ: Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子間力顕微鏡によるイメージングのための細菌の固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

生物工学

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

原子間力顕微鏡を用いて組換えDNAとタンパク質の複合体の可視化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

原子間力顕微鏡と内皮グリコカリックスの機械的性質の定量化

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

原子間力顕微鏡を用いた生細胞の機械的特性を測定する

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

トラクション力顕微鏡用の細胞培養表面近くのローカライズレポーター蛍光ビーズのための新しい方法

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子間力顕微鏡イメージングおよびサポートされている脂質二重層のフォーススペクトロスコピー

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

イン・シトゥバイオプロセスにおける単細胞形態のリアルタイム測定のための顕微鏡検査

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

早期変形性関節症の検出に対する原子間力顕微鏡の応用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百個の C.アルビカン 細胞に対するバイオ原子間力顕微鏡測定の自動化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

ライブセル上の近くの同時レーザー走査共焦点と原子間力顕微鏡(コンポカル)

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...