次のプロトコルは、メリーランド大学の制度的動物ケア、利用委員会、治験審査委員会で受け付けています。プロトコルに従う中央政府、州およびローカルの基準とガイドラインの定めるバイオ セーフティに関するメリーランド大学のポリシー。
1. 目のサンプルの郭清
<ol><li>
地元の食肉処理場や組織の銀行から目のサンプルを取得します。全体の眼球を取得した場合は、即座にレンズ、添付 zonules、硝子体を抽出します。 注: 下記詳細はブタと人間の目に適用されます。<ol>
<li>消毒手術ハサミ、鉗子を使用して、カットし、強膜を取り巻くすべての余分な組織を削除します。</li>
		<li>しっかりとその側に目を保持し、かみそりの刃を使用すると、目の角膜から 3 mm の側に沿って小切開。眼の中の硝子体に達している十分なカットの深さを確認します。</li>
		<li>はさみを使用して、慎重に目のまわりを切開に沿ってさらにカット.レンズを刺さない。代表的な画像を図 2に示します。</li>
		<li>一度目の外径がカットされて、鉗子を使った眼の後部組織を削除します。鉗子でレンズ、zonules、毛様体、および接続されている硝子体を分離します。代表的なイメージは図 2Bに示すように。</li>
		<li>はさみとピンセットを使って、レンズは MLS にフラットに置くことができるので余分な硝子体を削除します。 メモ: 角膜移植の場合、角膜のボタンは手術で使用され、世界の残りの部分は研究目的のために利用できます。ただし、この部分のグローブは、レンズ担架セットアップのティッシュの準備でまだ使用できます。後部が得られる場合にのみステップ 1.1.4–1.1.5 を実行のみです。</li>
	</ol>
</li>
	<li>すべて 15% の漂白剤溶液中 30 分間の後郭清を使用する機器を消毒します。</li>
</ol>2. トライアル アセンブリ マニュアル レンズ担架の
<ol><li>靴インデントの後ろの壁と靴自体の 5 mm 隙間が残っているので、MLS の底板に 10 mm の靴底と対応する靴トップを挿入します。</li>
	<li>上部と下部のプレートは、プレートを一緒にスナップを揃えるデバイスが今ばねの位置です。</li>
	<li>プレート ケースと底板の側面にある穴にストッパーのネジにプレートを挿入します。</li>
	<li>ベースにプレート ケースに挿入し、一直線に並べられたインデントにレンチを配置します。</li>
	<li>靴を元の伸縮位置に戻ります戻る反時計回りにひねるし、靴を契約する停止ネジに達するまでレンチを時計回りにねじる。</li>
</ol>3. レンズの装着
<ol><li>5 mm の差のまま靴インデントと靴自体の後ろの壁、MLS で底板に 10 mm の靴底を挿入します。</li>
	<li>底板の真ん中に抽出されたレンズは表向きに置きカーブタイプ鉗子を使用して、靴は、中央の穴にレンズを支えています。</li>
	<li>、クリッピングだけ zonules と硝子体に対応する靴の上部をはめ込みます。視覚的に、維持レンズ中心とした底板の可能な限り。</li>
	<li>手順 2.3-2.4.</li>
</ol>4. レンズの測定
<ol><li>ビデオやストレッチのプロセスの写真をキャプチャするために装置の上に直接イメージング システムを配置します。定規を事後処理で正確にサイズとスケール画像に画像のフレームに含めるようにしてください。 注: 任意の適切なイメージング システムはこの手順のため十分ですここでは、12 メガピクセル, オート フォーカス スマート フォンのサンプルから片方の足を使用します。</li>
	<li>しっかりとまだスムーズにレンズをストレッチする時計回りの方向にレンチを回転します。図 3では、ばねと伸ばされた状態で代表的なイメージを示しています。</li>
	<li>拡大レンズを撮影後、サンプルを安静時の状態に復元する反時計回りの方向にレンチを回転します。 注意: レンズの脱水症状を最小限にするために、タイムリーでレンズの測定を実行することが不可欠です。</li>
	<li>明らかに写真レンズの最終的な静止状態。</li>
</ol>5. データの解析
<ol><li>ImageJ に画像をアップロードし、「ポイント」機能を使用して、図 4に示すように、レンズのまわりを少なくとも 40 ポイントを選択します。「分析」→「メジャー」オプションを使用すると、各選択したポイントの位置をもたらします。</li>
	<li>(ソフトウェア例えばMATLAB を使用して) 半径とカイ二乗適合図 4Bに示すように生成するために場所という点に合います。ピクセル半径とエラーを撮影した定規を使用して測定値に変換します。</li>
	<li>2 尾 t 検定 MLS からストレッチの前後に個々 のレンズの比較を実行します。</li>
</ol>

<ol>
	<li>Von Helmholtz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+akkommodation+des+auges.">Uber die akkommodation des auges.</a> Arch Ophthal. 1, 1-74 (1855).</li><li>Schachar, R. A., Black, T. D., Kash, R. L., Cudmore, D. P., Schanzlin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+accommodation+and+presbyopia+in+the+primate.">The mechanism of accommodation and presbyopia in the primate.</a> Ann Ophthalmol. 27, 58-67 (1995).</li><li>Glasser, A., Campbell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Reilly, M. A., Hamilton, P. D., Perry, G., Ravi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+behavior+and+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior and natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88, 483-494 (2009).</li><li>Langenbucher, A., Huber, S., Nguyen, N. X., Seitz, B., Gusek-Schneider, G. C., K&#252;chle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+accommodation+after+implantation+of+an+accommodating+posterior+chamber+intraocular+lens.">Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.</a> J Cataract Refract Surg. 29 (4), 677-685 (2003).</li><li>Ehrmann, K., Ho, A., Parel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+analysis+of+the+accommodative+apparatus+in+primates.">Biomechanical analysis of the accommodative apparatus in primates.</a> Clin Exp Optom. 91 (4), 411 (2008).</li><li>Pinilla Cort&#233;s, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926 (2015).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastic+constants+of+the+human+lens.">The elastic constants of the human lens.</a> J Physiol. 212 (1), 147-180 (1971).</li><li>Eppig, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+eye+model+and+measurement+setup+for+investigating+accommodating+intraocular+lenses.">Biomechanical eye model and measurement setup for investigating accommodating intraocular lenses.</a> Z Med Ohys. 23 (2), 144-152 (2013).</li><li>Manns, F., Parel, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+and+Monkey+Lenses+in+a+Lens+Stretcher.">Response of Human and Monkey Lenses in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 48 (7), 3260 (2007).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li><li>Besner, S., Scarcelli, G., Pineda, R., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Brillouin+Analysis+of+the+Aging+Crystalline+Lens.">In Vivo Brillouin Analysis of the Aging Crystalline Lens.</a> Invest Ophthalmol Vis Sci. 57 (13), 5093 (2016).</li><li>Cortes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926-2932 (2015).</li><li>Bernal, A., Parel, J. -. M., Manns, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Posterior+Zonular+Fiber+Attachment+on+the+Anterior+Hyaloid+Membrane.">Evidence for Posterior Zonular Fiber Attachment on the Anterior Hyaloid Membrane.</a> Invest Ophthalmol Vis Sci. 47 (11), 4708 (2006).</li><li>Kammel, R., Ackermann, R., Mai, T., Damm, C., Nolte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+Lenses+in+a+Lens+Stretcher.">Pig Lenses in a Lens Stretcher.</a> Optom Vis Sci. 89 (6), 908-915 (2012).</li><li>Hahn, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Ex+Vivo+Porcine+Lens+Shape+During+Simulated+Accommodation,+Before+and+After+fs-Laser+Treatment.">Measurement of Ex Vivo Porcine Lens Shape During Simulated Accommodation, Before and After fs-Laser Treatment.</a> Invest Ophthalmol Vis Sci. 56 (9), 5332-5343 (2015).</li><li>D'Antin, J. C., Cortes, L. P., Montenegro, G. A., Barraquer, R. I., Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+portable+manual+stretching+device+to+simulate+accommodation.">Evaluation of a portable manual stretching device to simulate accommodation.</a> Acta Ophthalmol. 93 (255), (2015).</li><li>Pierscionek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Marussich, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Crystalline+Lens+Volume+During+Accommodation+in+a+Lens+Stretcher.">Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 56 (8), 4239 (2015).</li><li>Martinez-Enriquez, E., P&#233;rez-Merino, P., Velasco-Ocana, M., Marcos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OCT-based+full+crystalline+lens+shape+change+during+accommodation+in+vivo.">OCT-based full crystalline lens shape change during accommodation in vivo.</a> Biomed Opt Exp. 8 (2), 918-933 (2017).</li></ol>

AB は Bioniko コンサルティング LLC の持分です。

我々 は、サンプルに担架をカップルにデュアル部分クランプ機構を用いたレンズの収容能力を勉強の正確かつ効率的な方法を提供する手法を考案した.宿泊施設の中にレンズを緩和、直径はチン小帯1,2,4,19の緩和に対する応答で減少します。メソッドは、クランプし、チン小帯の緊張を制御するこ...

宿泊施設は、人間の目は鋭い焦点に遠くまたは近くの距離でオブジェクトを参照する、水晶体の形を動的に調整することができるプロセスです。宿泊施設は、本質的に生体力学的プロセスです。神経系の刺激には、毛様体筋は、レンズ カプセル1,2のまわりにアタッチ チン毛様体に力を生成します。宿泊施設のバイオメカニクスの背後にある別の理論がありますが、最も広く受け入れられているはヘルムホルツ仮説です。仮説によればレンズは、遠くのオブジェクトのフォーカスに最適なレンズの薄形に対応する自然な伸ばされた状態でです。毛様体筋の契約、近いオブジェクトにフォーカスを変更して、チン小帯が緩和されます。ターンでは、レンズが厚くなる、前部と後部の表面曲率の増加します。これは近くのビジョンは、したがってより短い焦点距離1に必要な屈折力の増加に対応しています。
老眼をという条件で時間をかけて対応する能力が損なわれます。老眼年齢 50 によって皆に影響を与える、目の距離3までいくからフォーカスを動的に変更することができませんになります。老眼に対処するため、現在の方法、矯正レンズ、遠近両用メガネなど受動的。いくつかの面で近いオブジェクトに焦点を当てる能力を増加しながらこのような受動的な治療はレンズ4、5のダイナミック フォーカス機能を復元できません。効率的に、老眼の治療、またはおそらくそれを防ぐため、継続的宿泊施設をよりよく理解する必要があります。
焦点調節を研究するには、4,6,7,8,9前のヴィヴォ現象をシミュレートするデバイスの数が開発されています。回転ディスクはまず遠心力8を介してレンズの伸縮を監視に導入されました。現象をより忠実に複製するには、レンズ伸縮装置が徐々 に導入し、革新。レンズ担架、に対するらを使用しています。レンズ電源と赤道の直径9そのような相関しながらレンズを収めるために必要な力を特徴とします。現在の理解は、レンズ硬直毛様体3,10、11,12の等しい力に応じ、レンズ形状の減少変更を伴う年齢とともにです。
現在レンズ担架ではしばしば、複雑なセットアップでは、エレクトロニクスおよびプログラム可能な伸縮率の実装を含む全体死体眼球6,7,10,13を必要とします。この要件は実験目あたり 500.00 ドル以上あたりのコストを増加し、サンプルの可用性が低下します。眼後部の合計約 $200.00 と低コストで焦点調節をレプリケートする方法をご紹介します。しばらくの間、今日使用される多くのデバイスよりもより少なく洗練された技術はよりコスト効果的で採用可能な結果を損なうことがなくです。このメソッドは、図 1に描かれているマニュアル レンズ担架 (MLS) を中心とし、レンズの直径を拡大する、チンと放射状のねじれ法に関するユニークなクランプ システムを使用しています。ベルナルらの調査結果によって、プロトコルの生理学的な精度を検証します。、前部と後部のチンがレンズ カプセル14を接続する経路を勉強しました。生理学的宿泊施設をレプリケートすることによってレンズのバイオメカニクスを研究するのみレンズ、チン、毛様体を必要とするカスタムの靴のデザインを使用して、目指しました。

<table><tbody><tr><td>Manual Lens Stretcher</td><td>Bioniko</td><td>MLS</td><td>Different animal species will require different shoe sizes</td></tr><tr><td>Porcine Eye Samples</td><td>George G. Ruppersberger; slaughterhouse</td><td>N/A</td><td>Whole eyeballs were obtained</td></tr><tr><td>Human Eye Samples</td><td>The National Disease Research Interchange</td><td>N/A</td><td>Posterior poles without corneas were ordered</td></tr><tr><td>Dissecting Scissors (5 1/2&#039;&#039; Straight)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>Tissue Forceps (4 1/2&#039;&#039;)</td><td>Electron Microsopy Sciences</td><td>72960</td><td></td></tr><tr><td>iPhone 6s</td><td>Apple</td><td>N/A</td><td>Any imaging system with ~0.1 mm resolution will work</td></tr><tr><td>Sodium Hypochorite</td><td>Clorox</td><td>Clorox Regular-Bleach</td><td>Any disinfectant will work</td></tr></tbody></table>

simulating the mechanics of lens accommodation via a manual lens stretcher

このプロトコルの目的は、コスト効率の高い、実用的な方法で生理学的宿泊施設の生体力学を模倣することです。宿泊施設は、毛様体の収縮と弛緩、チンの近見視力に必要なレンズの肥厚の結果によって実現されます。ここでは、小説、宿泊施設をレプリケートして、マニュアル レンズ担架 (MLS) を介して水晶体カプセルに接続されている zonules の緊張によって簡単な方法を提案します。このメソッドは一貫した力を受けるときのレンズにより橈骨ストレッチを監視し、ストレッチできません非対応のレンズに、伸ばすことができるレンズを収容を比較できます。重要なは、ストレッチャー カップル zonules を直接していない地球全体のサンプルではなく、レンズ、zonules、および毛様体従ってだけ必要とする、目の強膜。この違いは、地球全体の取得と比較して約 62% の死体ドナーのレンズを取得のコストを減らすことができます。

ブタ目、ストレッチ4,15、レンズによる老眼を勉強するための一般的なサンプルが得られた (n = 10) ローカル屠殺場、このプロトコルからレンズの収容能力を観察するため。図 5Aは MLS を介してストレッチの前後にブタのレンズの比較を示しています。拡大レンズの半径の平均 0.19 ± 0.07 mm 増...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

マニュアル レンズ担架による焦点調節機構のシミュレーション

マニュアル レンズの担架を使用して焦点調節を勉強の効率的な方法を提案します。プロトコル mimics 生理宿泊施設、zonules を引いてレンズ カプセル周りにより、接続されているレンズをストレッチします。

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

simulating-mechanics-lens-accommodation-via-manual-lens

Research

JoVE Journal

Bioengineering

6.7K ビュー.  University of Maryland. マニュアル レンズの担架を使用して焦点調節を勉強の効率的な方法を提案します。プロトコル mimics 生理宿泊施設、zonules を引いてレンズ カプセル周りにより、接続されているレンズをストレッチします。

ビデオ: Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

コンスタント·フォース軸光ピンセットを用いたDNAの短い配列を伸ばす

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

生物工学

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS膜上のマイクロパターン細胞を伸ばす

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

細胞および組織メカノでのアプリケーションのためのプラットフォームをストレッチ小説

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

メカニカルストレッチ中のライブセルイメージング

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

眼内レンズ用ソフト ポリシロキサン尿素エラストマーの合成

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

化学

Production of a Strain-Measuring Device with an Improved 3D Printer

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in the analog electrical signal in a traditional strain gauge operation, a new strain measurement method is presented here. It uses a photographic technique to display the strain change by amplifying the change of the pointer displacement of the mechanism. A visual polydimethylsiloxane (PDMS) lens with a focal length of 7.16 mm was added to a smartphone camera to generate a lens group acting as a microscope to capture images. It had an equivalent focal length of 5.74 mm. Acrylonitrile butadiene styrene (ABS) and nylon amplifiers were used to test the influence of different materials on the sensor performance. The production of the amplifiers and PDMS lens is based on improved 3D printing technology. The data obtained were compared with the results from finite element analysis (FEA) to verify their validity. The sensitivity of the ABS amplifier was 36.03 &plusmn; 1.34 &#181;&epsilon;/&#181;m, and the sensitivity of the nylon amplifier was 36.55 &plusmn; 0.53 &#181;&epsilon;/&#181;m.

This work presents a strain measurement sensor consisting of an amplification mechanism and a polydimethylsiloxane microscope manufactured using an improved 3D printer.

改良された3Dプリンターを用いた歪み測定装置の製造

A traditional strain measurement sensor needs to be electrified and is susceptible to electromagnetic interference. In order to solve the fluctuations in ...

工学

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

ライブ顕微鏡イメージング下での3Dヒドロゲルの制御株

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

自動圧縮によるブタ眼レンズの生体力学的特性評価

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

著者スポットライト:加齢に伴うレンズの剛性変化の理解を深める 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

接眼レンズの自動圧縮試験

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

マニュアルレンズ担架による焦点調節機構のシミュレーション

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マニュアル レンズ担架による焦点調節機構のシミュレーション

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マニュアルレンズ担架による焦点調節機構のシミュレーション