この作品が支持されたオーストリアのアカデミー科学 (CTH) のアパートの付与、チェコ共和国の技術振興プロジェクトなし。TA04010877 (CTH、VH と国債)、およびチェコ科学財団 (GACR) プロジェクト (国債) に GA16 01246S のないです。義歯定着性のクリームを使用する彼の提案のためのテカムセ フィッチ w. と Ing に感謝しますP. リシュカ摘出鹿 larynges を取得するのに彼の助けをチェコ軍のフォレスト サービスから。

本稿で分析した動物の標本は、パラツキー大学オロモウツ、チェコ共和国での標準倫理的な要件に従って扱われました。彼らは赤いシカの通常の狩猟シーズン中にチェコ軍森林サービスによって捜された林で乱暴生活から生じる。
1. 片側喉頭標本の準備
注: 唯一の適切に準備された標本使わなければなりません、4に示すように。喉頭の凍結迅速な切除し-80 ° C で保存した直後に25組織分解の潜在性と力学的特性の変化を最小限に抑えるし、でいつでも実験を実行することができます。
<ol><li>
喉頭を解凍
	<ol>
<li>2 オートクレーブ バッグや防水シールと他のビニール袋に冷凍の喉頭を挿入します。袋を密封して喉頭を完全に解凍するまで 30 の ° C に加熱水のお風呂に入れます。必要な期間の範囲は、数時間から喉頭サイズおよび凍結温度によってより多くの日。</li>
	</ol>
</li>
	<li>
喉頭をクリーニング
	<ol>
<li>バッグから削除し、徹底的にそれをきれいに生理食塩液を喉頭を解凍後 (0.9 %nacl)。</li>
		<li>慎重に必要に応じて、余分な組織を削除 (すなわち外部の首の筋肉、舌骨など) なしメインの喉頭の構造の損傷や気管、空気供給管に喉頭をマウントするために十分な長さを短縮 (ca. 通常 4-5 cm)。</li>
		<li>潜在的実験には不向き、喉頭を作ることができる凍結の過程から発生するクラックや有機の変形、傷などの潜在的な生体組織異常のため喉頭組織を確認します。</li>
	</ol>
</li>
	<li>
甲状腺と輪状軟骨の露出
	<ol>
<li>正中カット片側喉頭の作成の準備で軟骨を公開するので、メスを用いた甲状腺と輪状軟骨周囲の外喉頭筋組織の部分を削除します。この準備段階は、図 1 aおよび1 bで描かれています。</li>
	</ol>
</li>
	<li>
正中甲状軟骨を切ってください。
	<ol>
<li>甲状軟骨の前部の初期垂直カットを作る。</li>
		<li>保存状態を維持する必要がある声帯に損傷を与えるために、削除される側に少しカットして慎重に置きます。可能であれば、切断用メスを使用します。軟骨が骨化すると場合、は、小さな鋸を使用します。</li>
	</ol>
</li>
	<li>
輪状軟骨を切断
	<ol>
<li>垂直方向にカットをリード (下方に) から披裂軟骨の間に放置して下甲状腺ノッチの約水平レベルに輪状軟骨。</li>
	</ol>
</li>
	<li>
喉頭に L 字切開を作成する 1 つの声帯除去
	<ol>
<li>輪状軟骨で、以前作った垂直カットの下の端から始まってを切り取って、下甲状腺切り込みに向かって新しいカットを導く水平を確認します。前方削除する起こっている喉頭の側面を隠します。</li>
		<li>甲状軟骨の内側の軟部組織を切って、甲状軟骨、声帯への損傷を避けるために、声帯の前方の添付ファイルの間にカットをリードしながら注意してください - 垂直を確認します。</li>
	</ol>
</li>
	<li>
甲状軟骨のカットの絞り込み
	<ol>
<li>以前検査した声帯の前方部分にできるだけ近いメス、のこぎり、または甲状軟骨で正確にストレート カットを適用し、として取得するために、ファイルを使用します。</li>
		<li>また後部の甲状軟骨の一部を削除、披裂の内転の芯を挿入するためのスペースを作成するために軟骨そしてこうしてボーカル折り (下記参照)。この準備段階は、図 1と1 Dで描かれています。 注: 研究の質問によって声帯全体における博覧会は上からの表示を有効にするのに必要かもしれない。このような場合は、(真) の声帯 (すなわち、心室または前庭倍、解剖学標本の指定に応じて) 上の構造を削除してください。いくつかの標本は声帯より上内側の軟喉頭部甲状軟骨との接続を失う可能性があり、声帯振動時に干渉、スプリアスの (大抵不規則な) 振動パターンを引き起こして可能性があります。このようなケースでは、その組織を慎重に除去は避けられないです。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55303/55303fig1.jpg"> 図 1: ヘミ喉頭準備および取り付け。(A) と (B) クリーニング喉頭片、左声帯; 削除する前に、内側と後部のビュー(C) と (D) 準備されたヘミ喉頭 L 字切開 (左声帯除去)、内側と後部のビュー。<a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

2. 片側喉頭実験
<ol><li>
ヘミ喉頭セットアップ
	<ol>
<li>喉頭に温め、加湿空気を提供する空気供給管を使用します。</li>
		<li>構造 2 は垂直に削除された喉頭部分の代替として透明板を配置しました。</li>
		<li>使用熊手が喉頭の安定性が向上し、縦のガラス プレートに残りの声帯の内転によって適切な pre-phonatory 喉頭構成を作成するため4 (図 2 a参照)。 注: 理論的には、声帯可能性がありますもが内転し縫合糸と滑車レバー システム26の重みで。しかし、このようなアプローチは、これらの著者の最高の知識にまだ試みられて hemilarynx 準備のため。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55303/55303fig2.jpg">
図 2: ヘミ喉頭セットアップします。(A) 支持構造: 空気供給管、L 型のガラス プレート アレンジ、内転の熊手。(B) 片側喉頭内転の突起を持つ準備をマウントします。(C)、ヘミ-喉頭-準備の (D) クローズ アップ表示側からと、上からそれぞれ。<a href="//cloudflare.jove.com/files/ftp_upload/55303/55303fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"><li>
ヘミ喉頭をマウント
	<ol>
<li>義歯固定剤クリームを空気供給管をカバーし、喉頭、気管の残りの部分をマウントします。定着性のクリームは接着剤として働き、従って気密のシールを作成する、潜在的なギャップを閉じます。</li>
		<li>プラスチック製のストラップやホースのクランプを締めと気管を固定します。</li>
		<li>また定着剤の定着性声帯や喉頭生体組織の内部ソフト クリームを拡散を回避しながら、クリームと甲状軟骨のカットのエッジをカバーします。</li>
		<li>透明の板を取り付けます。</li>
	</ol>
</li>
	<li>
甲状軟骨の突起を使用して声帯の内転の安定化
	<ol>
<li>付加体をプレートに声帯、甲状軟骨を安定させる突起を使用します。</li>
		<li>定着性のクリームが設定後は、声帯振動とヘミ喉頭とガラス板の可能なリークのチェックを確立するために空気の流れを適用します。</li>
		<li>多く定着性のクリームを追加することによって、最終的に発生するギャップをシールします。</li>
	</ol>
</li>
</ol>

著者が明らかに何もありません。

ヘミ喉頭準備を共有する「従来型」の (完全な) 摘出喉頭セットアップの利点: このような実験的アプローチ、物理的、生理的な境界条件とパラメーター (声門圧力や声帯伸長) などはすることができますかなりよく制御されます。Hemilarynx の動作は相同性と完璧な完全喉頭の横方向の対称性、例外とその大きさのいくつかのパラメーターの (e.g、風量、音圧) 約 50% 削減はまだまだ内にあ?...

声は、空気パルス シーケンスに肺によって提供される安定した気流を変換する喉頭組織 (主に声帯ヒダ) を振動によって通常作成されます。音圧波形 (つまり、プライマリの音)、それらをフィルタ リングする声道音響流パルスこのシーケンスから新興興奮させるし、鼻1 から放射口からと (ある程度) に結果としての音.生成された音のスペクトル構成主、声帯振動、喉頭のバイオメカニクスと2気管気流との相互作用によって支配の質によって影響されます。臨床と研究のコンテキストの両方で、ドキュメントと声帯振動の評価は、何よりも興味の発声を学ぶとき。
ヒトでは、体内の健全な生産の間に喉頭の直接内視鏡調査は挑戦的なとは現在技術的な手段を与えられるヒト以外の哺乳類のほぼ不可能です。したがって、慎重に制御保証物理的および/または生理学的な実験的境界条件、摘出 larynges3,4の使用する順番は、多くの場合の調査のための適切な置換体内音声生成機構。
声帯振動は、複雑な 3次元現象5です。従来の調査法のような (体内) 喉頭内視鏡検査または摘出喉頭準備通常ビューを提供のみ優れた声帯の振動6、彼らはの完全な三次元解析を許可しません。声帯運動。特に、優れたビューで声帯の下 (尾) 余白は見えない振動サイクルの主要な部分の間に。これは劣る (尾) と (頭蓋) 縁声帯、声帯振動5中に通常見られる現象間の位相遅延が原因です。数理モデルから調査結果をバックアップするための直接経験的な証拠は乏しい、ジオメトリの知識と低い声の動きを折るエッジ7、およびこうして声門チャネル8,9のジオメトリ,10は喉頭・声帯組織・結果の荷重や圧力11,12間の相互作用の理解のために重要です。恒例の優れたビューから隠されている声帯振動の別の側面は、2 つの声帯間の接触の垂直 (caudo 頭蓋) 深さです。連絡先奥行き歌 (レジスタ「裏声」対「胸」)13,14で使うボーカル レジスタの潜在的な指標は、声帯ヒダの縦の厚さに関連しています。
ここで 1 つの半分は、いわゆる片側喉頭セットアップ従来 (フル) 摘出喉頭製剤の欠点を克服するために利用できる喉頭が削除されるため、残りの振動特性の評価が容易3 つの次元で声帯。驚いたことに、この 1960 年代15設定の導入、1993年16における概念の初期検証多くない所はこの有望な実験的アプローチと17,18 実験を実行しています。 ,,1920,21,22,23。これのための説明は、実行可能な片側喉頭準備の作成の難しさの発見可能性があります。従来の切除 (フル) 喉頭の準備は十分に文書化4が、このような詳細な手順がないまだ片側喉頭のセットアップを作成するため。したがって、このペーパー レッド ディアの標本から実験結果によって補われる確実に再現可能な片側喉頭セットアップを確立するためのチュートリアルを提供する目的です。
ヘミ喉頭セットアップは測定装置、高速などの「従来型」の摘出喉頭セットアップまたはサウンドの生成中または適切な喉頭の構造物の振動を適切に文書に他のイメージング技術と多くの機能を共有します。加熱、加湿空気の供給。これらの一般的なセットアップの考慮事項は、本章4国立センターの音声、音声24から技術的なレポートで詳細に説明します。この原稿の範囲を超えてこれらの指示の繰り返しになります。ここでは、ヘミ喉頭セットアップを生成するため特殊なディレクティブのみが掲載されています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Surgical blades</td>
			<td >Surgeon</td>
			<td >Jai Surgical Ltd., New Delhi, India</td>
			<td ></td>
		</tr>
		<tr >
			<td >Saw</td>
			<td >Hand saw (Lux, 150 mm length)</td>
			<td >Lux, Wermelskirchen, Germany</td>
			<td ></td>
		</tr>
		<tr >
			<td >Thermometer</td>
			<td >Testo 922</td>
			<td >Testo Ltd., Hampshire, UK</td>
			<td >K-type Probe, Operating temperature -20 to +50 &#176;C</td>
		</tr>
		<tr >
			<td >Autoclave bags</td>
			<td >Autoclave bags</td>
			<td >vwr.com, VWR International s.r.o., Stribrna Skalice, Czech republic</td>
			<td ></td>
		</tr>
		<tr >
			<td >Conductive glass plates</td>
			<td >Custom made</td>
			<td >UPOL - Joint laboratory of Optics 
			Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fixative cream</td>
			<td >Denture fixative cream</td>
			<td >Blend-a-dent Natural</td>
			<td ></td>
		</tr>
		<tr >
			<td >Prongs and fastening system</td>
			<td >Customized Kanya Al eloxed profiles</td>
			<td >Distributor: VISIMPEX a.s.. Seifertova 33, 750 02 Prerov, the Czech Rep.;</td>
			<td >&#160;Combination of Kanya RVS and PVS fastening systems (http://www.kanya.cz/) + custom made prongs</td>
		</tr>
		<tr >
			<td >Mounting tube</td>
			<td >Custom made</td>
			<td >UPOL - Joint laboratory of Optics, 
			Trida 17. listopadu 50A, 772 07 Olomouc, the Czech Rep.</td>
			<td ></td>
		</tr>
		<tr >
			<td >LED Light</td>
			<td >Verbatim 52204 LED Lamp</td>
			<td >Mitsubishi Chemical Holdings Corporation, Tokyo, Japan</td>
			<td ></td>
		</tr>
		<tr >
			<td >Camera</td>
			<td >Canon EOS1100D</td>
			<td >Canon Inc.</td>
			<td >18-55 mm lens</td>
		</tr>
		<tr >
			<td >Airpump</td>
			<td >Resun LP100</td>
			<td >Resun</td>
			<td ></td>
		</tr>
		<tr >
			<td >Strobe light</td>
			<td >ELMED Helio-Strob micro2</td>
			<td >ELMED Dr. Ing. Mense GmbH, Heiligenhaus, Germany</td>
			<td ></td>
		</tr>
		<tr >
			<td >Humidifier</td>
			<td >Custom made</td>
			<td >Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td>
			<td ></td>
		</tr>
		<tr >
			<td >Subglottic tract</td>
			<td >Custom made adjustable subglottic tract</td>
			<td >Voice Research Lab, Dept. Biophysics, Faculty of Sciences, Palacky University Olomouc, Czech republic</td>
			<td >Hampala, V., Svec, Jan, Schovanek, P., and Mandat, D. Uzitny vzor c. 25585: Model subglotickeho traktu. [Utility model no. 25585: Model of subglottal tract] (In Czech) Soukup, P. 2013-27834(CZ 25505 U1), 1-7. 24-6-2013. Praha, Urad prumysloveho vlastnictvi</td>
		</tr>
	</tbody>
</table>

hemi-laryngeal setup for studying vocal fold vibration in three dimensions

ほとんどのヒト以外の哺乳類と人間の声は、声帯の自立振動を通して喉頭で生成されます。声帯振動の直接の映像記録は、ヒト以外の哺乳類で特に困難です。別の方法としては、摘出喉頭実験は管理された生理学的および物理的な条件の下で声帯振動を調査する機会を提供します。ただし、完全喉頭の使用は単空気力との相互作用観測による振動の構造の重要な部分を除く、声帯の平面図を提供します。ここで 1 つの半分にヘミ喉頭セットアップを利用してこの制限を克服できる喉頭の半ば矢削除、自励発振時に優れたと残りの声帯の横方向ビューを提供します。
ここでは、研究室のベンチに片側喉頭の構造と、取り付けの解剖学的な準備のためのステップ バイ ステップ ガイドが与えられます。ヘミ喉頭準備の模範的な発声は、2 つの同期カメラ (スーペリアーと横方向の眺め)、示す三次元声帯運動および対応する接触面積の時間変化を取り込んだ高速ビデオ データを記載されています。このパブリケーションでヘミ喉頭セットアップのドキュメント アプリケーションと再現性の実験的研究、発声のバイオメカニクスを理解する可能性のある音声科学者を提供することで容易になります。

ヘミ喉頭準備をし、前のセクションで参照している、空気供給管に取付部のイラストは、それぞれ図 1と図 2で提供されます。
2 つのカメラ アングルから声帯振動のドキュメント
片側喉頭の声帯の気流力自立振動上から...

Watch this Scientific Journal Video about Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions at JoVE.com

Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

本稿ではヒトとヒト以外の哺乳類における発声の様々 な生物物理学的側面を調査するために声帯振動の多次元ビューを促進するヘミ喉頭標本の準備のためのプロトコル。

3 つの次元で声帯振動を勉強のため片側喉頭セットアップ

The voice of humans and most non-human mammals is generated in the larynx through self-sustaining oscillation of the vocal folds. Direct visual ...

hemi-laryngeal-setup-for-studying-vocal-fold-vibration-three

Research

JoVE Journal

Biology

10.9K ビュー.  Palacky University Olomouc. 本稿ではヒトとヒト以外の哺乳類における発声の様々 な生物物理学的側面を調査するために声帯振動の多次元ビューを促進するヘミ喉頭標本の準備のためのプロトコル。

ビデオ: Hemi-laryngeal Setup for Studying Vocal Fold Vibration in Three Dimensions

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

灌流マイクロバイオリアクターのチップベースの三次元細胞培養

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

FRET Imaging in Three-dimensional Hydrogels

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

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

三次元ハイドロゲルでイメージングをFRET

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

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

カポジ肉腫ヘルペス ウイルスによる細胞変換を勉強するための in Vitroモデル

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

の栽培<em&gt;線虫（Caenorhabditis elegans）</em&gt;ラボにおける三次元で

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

4倍のチェッカーボード:薬物の組み合わせを研究するための三次元チェッカーボードの変形

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

細胞外小胞の直接確率的光学再構成顕微鏡

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Studying Habituation in Stentor coeruleus

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display intelligent behaviors. In both natural and artificial systems, learning is the adaptive updating of system parameters based on new information, and intelligence is a measure of the computational process that facilitates learning. Stentor coeruleus is a unicellular pond-dwelling organism that exhibits habituation, a form of learning in which a behavioral response decreases following a repeated stimulus. Stentor contracts in response to mechanical stimulation, which is an apparent escape response from aquatic predators. However, repeated low-force perturbations induce habituation, demonstrated by a progressive reduction in contraction probability. Here, we introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency, including methods for building the apparatus and setting up the experiment in a way that minimizes external perturbations. In contrast to the previously described approaches for mechanically stimulating Stentor, this device allows the force of stimulation to be varied under computer control during the course of a single experiment, thus greatly increasing the variety of input sequences that can be applied. Understanding habituation at the level of a single cell will help characterize learning paradigms that are independent of complex circuitry.

Studying Habituation in Stentor coeruleus

We introduce a method for quantifying Stentor habituation using a microcontroller board-linked apparatus that can deliver mechanical pulses at a specified force and frequency. We also include methods for assembling the apparatus and setting up the experiment in a way that minimizes external perturbations.

ステントル・コエルレウスの馴化の研究

Learning is usually associated with a complex nervous system, but there is increasing evidence that life at all levels, down to single cells, can display ...

3 つの次元で声帯振動を勉強のため片側喉頭セットアップ

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灌流マイクロバイオリアクターのチップベースの三次元細胞培養

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

三次元ハイドロゲルでイメージングをFRET

カポジ肉腫ヘルペスウイルスによる細胞変換を勉強するための in Vitroモデル

の栽培

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

4倍のチェッカーボード:薬物の組み合わせを研究するための三次元チェッカーボードの変形

細胞外小胞の直接確率的光学再構成顕微鏡

ステントル・コエルレウスの馴化の研究

3 つの次元で声帯振動を勉強のため片側喉頭セットアップ

要約

さらに動画を探す

この動画の章

灌流マイクロバイオリアクターのチップベースの三次元細胞培養

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

三次元ハイドロゲルでイメージングをFRET

カポジ肉腫ヘルペス ウイルスによる細胞変換を勉強するための in Vitroモデル

の栽培

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

4倍のチェッカーボード:薬物の組み合わせを研究するための三次元チェッカーボードの変形

細胞外小胞の直接確率的光学再構成顕微鏡

ステントル・コエルレウスの馴化の研究

カポジ肉腫ヘルペスウイルスによる細胞変換を勉強するための in Vitroモデル