著者は、博士に感謝したいと思います。和平チェン、Huiliang張とスティーブンKolwiczこの方法を開発する上で有益なコメントや技術的なサポートのため。この研究は、NIHの助成金や米国心臓協会からWWに科学者開発助成金によって支えられている。

1。実験の準備<ol><li> 140mMのNaCl、5mMのKCl、2.5mMのCaCl 2、2mMのMgCl 2および10mM HEPES インビボでの骨格筋イメージング用液（pH 7.2）を含有する等張平衡塩類溶液（50ml）に調製する。 </li><li> 118のNaCl、5.3のKCl、1.2のMgSO 4、0.5mMのEDTAおよび25mMの炭酸水素ナトリウムを ：含むクレブス-ヘンゼライト緩衝液（KHB）を1リットル調製。 </li><li>従来2mMのCaCl 2を添加する10〜15分間、95％O 2/5％CO 2を含むガスと気泡KHB。代謝基質（ 例えば 、10 mMグルコースおよび0.5mMのピルビン酸）を追加します。 </li><li> 70％エタノールでクランプ、はさみ、鉗子を滅菌した後、滅菌蒸留H 2 Oにすすぐことにより手術器具を準備します</li><li> 、心臓の灌流システムの電源を入れ、水浴、サーキュレータの温度を調整し、6ミリリットル/分に蠕動ポンプの速度を設定します。 </li><li> 95％O 2と気泡KHB流出物/ 5％CO 2ガス、および（光ファイバ温度プローブによって37℃）、流速および温度を監視する。システムは、ガスと温度平衡化のために約20分を必要とします。 </li></ol> 2。 in vivoでの骨格筋の共焦点イメージング<ol><li>ペントバルビタール（80 mg / kgで、腹腔内）でマウスを麻酔。動物は、10〜15分以内に、手術の麻酔（つま先のピンチに応答なし）に到達し、骨格筋のin vivoイメージングのために十分な1〜1.5時間、この状態のままになります。 </li><li>後肢の1に毛を削除し、70％エタノールで皮膚を消毒する。 </li><li>腓腹筋を露出させ、肢の外側に沿って皮膚に切開する。 </li><li>鋭いピンセットで軽く筋外膜をピックアップし、ハサミでそれを介して切開する。さらに筋外膜を取り外し、その下の筋線維を露出するために解剖。 </li><li>骨格へのTMRM のin vivoロード用筋肉は、30分間の筋肉を浸した後、指示薬を含まない溶液で洗い流すこと等張平衡塩類溶液中のTMRM（500 nM）を含める。 </li><li>共焦点顕微鏡（Zeiss LSM 510）ステージ上で横にマウスを置くと、公開される骨格筋は、チャンバーの底部を形成するカバーガラスに対向する位置に、リア手足を拘束。カバースリップは、筋肉組織および反転対物レンズ（40X油）との間にある。 </li><li>組織とカバーガラスの間に密着させるようにそっと足を押してください。等張平衡塩溶液中で公開される筋肉を浸す。 </li><li>フレームあたり1秒のサンプリングレートで録音する2次元（2D、 メトキシ）共焦点画像。各画素の強度は、8ビットの深度でデジタル化される。通常、シリアルスキャンは100フレームが含まれています。 </li><li> &gt; 505 nmで最初の405 nmでのエキサイティングで収集することにより連続した画像を収集し、488 nmの二波長excitatio用&gt; 505 nmで収集しながら、MT-cpYFPのNイメージング。 MT-cpYFPとTMRMの三波長励起イメージングのために、それぞれ、405、488および543 nmで連続励起を使用し、505から545の発光を収集し、505から545、および&gt; 560 nmの。 </li></ol> 3。灌流マウス心臓の共焦点イメージング<ol><li>すぐに骨格筋のin vivoイメージングの後、ヘパリン200単位（IP）でマウスを注入します。 10分後、開胸術により、マウスを安楽死させるとすぐにそれに接続されている肺や胸腺で心を取り除く。 </li><li>すぐに氷冷緩衝液で肺を削除します。胸腺の葉を識別し、ゆっくりと上行大動脈を露出するために戻ってはがします。 </li><li>胸腺を取り外し、慎重に周囲の組織を除去することにより、大動脈を隔離。 </li><li>大動脈弓の最初の分岐の前に、上行大動脈の上端の切断を行う。 </li><li>ルーメンを露出するために2マイクロ縫合鉗子で穏やかに大動脈の壁を持って、慎重にCAに大動脈を配置nnula（PE50チューブ）。縫合糸が迅速に大動脈の周りに縛られている間は、マイクロ大動脈血管クランプで所定位置に保持される。 </li><li>カニューレの先端が大動脈基部より上であることを確認するために鉗子でカニューレをチェックし、慎重に、クランプを取り外します。追加の関係を所定の位置に心臓を保持するために必要に応じて添加される。 </li><li>蠕動ポンプの電源を入れ、1ミリリットル/分で心臓を灌流。心臓は、灌流の際に暴行を再開します。 </li><li>灌流システムの位置を調整し、顕微鏡ステージ上で心を置く。ステージは、心を保持しているチャンバーの加熱を可能にするアダプタを持っています。 </li><li>部分的に心を沈めるためにチャンバー内KHB灌流溶液1mlを加える。 37℃のチャンバーの温度を監視する蠕動ポンプを使用することにより、チャンバからの流出物を除去します。 </li><li>心臓への十分な流量（約2ミリリットル/分）を提供するために、徐々に蠕動ポンプの速度を増加させる。 </li><li>安定化の10分後、ょんを潅流10μMのブレビスタチンおよび100〜500nmのTMRMとRT。ハートビートは10分後に遅くなります。 </li><li>チャンバーの下部にあるカバーガラスで心​​の緊密な接触を確実にするために、さらにハートビートを抑制するために心に静かに押し込みます。 </li><li>上記のステップ2.8で説明したように完全な心筋の共焦点イメージングのためのと同じ手順に従ってください。慎重に可能な鮮明な画像を明らかにするために、焦点面を調整します。 </li></ol> 4。画像処理およびデータ分析<ol><li>シングルミトコンドリアが点滅し、膜電位変化を分析するために、共焦点ソフトウェアの「生理的分析」モジュールによって提供されるツールを使用してください。このモジュールは、多くの場合、共焦点システムの画像取得ソフトウェアに含まれる画像の中の特定の領域の決定だけでなく、時間ラベルとの蛍光強度の出力を可能にする。 </li><li>データベースと分析しようとする、シリアル2D画像ファイルを開きます。 </li><li> 「地域をクリックしてください関心領域（ROI）は、平均ROIの &quot;に切り替える」という意味の平均ROIの「モード」に切り替え、「関心領域（ROI）は、平均&quot;モード。をクリックして &quot;。 </li><li>点滅を選択するためのcpYFP 488nmのチャンネルを除く他のチャンネルの表示をオフにします。 </li><li>ズーム画像と手動シリアル2D画像を再生するために、スライドバーを移動します。 </li><li>蛍光シグナルが一過性に増加し、サイトを配置することにより、単一のミトコンドリアのスーパーオキシドが点滅を識別します。点滅をマークするために、適切なROIツールを使用します。各ROIの時間依存蛍光変化を示すトレースは、画像の横に表示されます。 </li><li>すべての点滅を選択した後、他のチャンネルの表示をオン。バックグラウンド信号の減算の​​ための細胞の外側で画像にROIを選択します。一緒に時間ラベルで出力各ROIの平均蛍光を。 </li><li>のスキャン時間と面積と一緒にシリアルスキャンされた画像ファイルのそれぞれに点滅回数を記録しますセル。 Excelを使用して100秒/ 1,000μm2のセル面積あたりの点滅回数などのフラッシュ頻度を計算する。 </li><li>各フラッシュ（振幅、ピークまでの時間と減衰時間）のパラメータを計算するために対話型のデータ言語（IDL、ResearchSystems）で記述されたカスタム開発されたプログラムを使用してください。 IDLのソフトウェアは市販されており、フラッシュパラメータ解析用のカスタム開発されたプログラムは、要求に応じて作成者から得ることができます。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

生きた動物または灌流さの臓器の単一ミトコンドリアのイベントを画像化することは、ミトコンドリア機能評価17,19,21,22,24,25のための従来の方法に比べて大きな利点があります。ここに記載された技術は、細胞内の解像度で、実際の生理学的状態にあるミトコンドリア機能のその場での決定に 、リアルタイムを実現することができます。全身的にインビボで特定?...

ミトコンドリアは、細胞の生体エネルギー、フリーラジカルシグナル伝達、酸化還元ホメオスタシス、イオン調節、および細胞運命決定の1,2において中心的な役割を果たす。ミトコンドリア機能障害は、多くの場合、付属のと病気3-6の病因の基礎となる。特に、心臓や骨格筋などの筋肉のシステムでは、ミトコンドリアの呼吸は、細胞内カルシウム、堅牢な力開発7,8のタイムリーな調整を支援するために、ATPの大部分を提供します。これらの筋肉は、多くの場合、全細胞容積20〜40％まで占め、筋フィラメント2間に「固定」されたミトコンドリアの多数を持っている。 多くの研究にもかかわらず、ミトコンドリア機能調節の我々の理解は、特に生体内および生理学的に関連する条件下では、限られている。理由の一つは、ミトコンドリア機能を評価するために開発された方法の大部分はvitrに頼るあるこのような人工基質を補充した単離ミトコンドリアの酸素消費量、および間接形態学を通じてミトコンドリア機能の決定（ 例えば電子顕微鏡）、酵素活性（ 例えばアコニターゼ活性）、または細胞内ATPレベル9から11の監視などoまたはex vivoでのアプローチ、 。 最近では、相対的なミトコンドリア富化を有する小分子蛍光指示薬は、無傷の細胞における膜電位、カルシウムおよび活性酸素種（ROS）、11-13を含むミトコンドリアシグナルの姿を提供するために適用されている。さらに、いくつかの緑色蛍光タンパク質（GFP）をレドックス系、ROS指標区画化、細胞内の酸化還元またはROS 14-16信号のより具体的な評価を達成するために開発されてきた。この中でも、我々は、遺伝的にコード化されたスーパーオキシドインジケーター、円形の順列黄色蛍光タンパク質、およびtargeteを開発Dそれミトコンドリア（MT-cpYFP）17に変換する。 MT-cpYFPは、515 nmでの発光ピークの両方で405又は488nmで励起することができる。 488nm励起での発光は、 インビトロおよびインビボ較正 17,18 内の前で示すように、スーパーオキシドに特異的に応答する。 405 nm励起の発光は、（様々な条件の下で、MT-cpYFPの発光および励起スペクトルの詳細については文献[17の図1をご参照ください）​​内部コントロールとして使用されます。タイムラプス共焦点イメージングでは、この指標は、無傷の細胞の単一ミトコンドリア内スーパーオキシド産生のイベント、というスーパーが点滅し、破裂を検出。スーパーフラッシュは、ミトコンドリア呼吸、添付過渡ミトコンドリア膜脱分極およびROS産17〜20の合成関数として機能します。最近では、C57/BL6の背景をpUC-CAGGS-MT-cpYFPベクトル17,19を使用したパン-組織MT-cpYFPトランスジェニックマウスを作製し、強力なEXPRESを確認しました心臓、骨格筋および他の組織（ 図2）で、この指標のシオン。トランスジェニックマウスは、要求とワシントン大学のMTAの承認を得て興味を持って、学術研究者のために利用できるようになります。 本研究では、 その場でのランゲンドルフ灌流し、心臓内のスーパーオキシドが点滅の撮影だけでなく、麻酔MT-cpYFPトランスジェニックマウス17,19の骨格筋におけるFlashイベントのin vivoイメージングに説明します。この技術は、生理学的に関連する状態またはin vivo 21,22の単一ミトコンドリアのROS産生のイベントのリアルタイム監視が可能になります。このような適切な蛍光指示薬を有する膜電位およびカルシウムなどの他の単一ミトコンドリアのパラメータを監視するシステムを使用することも可能である。細胞内のイベント（ 例えば 、カルシウムトランジェント）や心臓の機能（ 例えばとミトコンドリア機能の更なる、同時またはパラレル評価。駆出率）を達成することができる。例えば、虚血および再灌流のような病理学的摂動は、無傷心筋の単一ミトコンドリア機能に対するストレスの影響を評価するために灌流心臓に適用することができる。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Toronto Research Chemicals</td>
			<td>B592500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros Organics</td>
			<td>AC34961-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7006-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1880-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256-25</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Invitrogen</td>
			<td>T-668</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &#34;Physiological Analysis&#34; module.</td>
		</tr>
	</tbody>
</table>

confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo

ミトコンドリアは、真核生物系でのエネルギー生産と細胞内シグナル伝達を担う重要な細胞内小器官である。ミトコンドリア機能障害は、多くの場合、付属の、人間の病気に貢献しています。ミトコンドリア機能および機能不全を評価するために開発されてきたアプローチの大部分は、in vitroまたはex vivoで測定に基づいている。これらの実験からの結果は、in vivoでのミトコンドリア機能を決定する上での能力が限られている。ここでは、 インビボでリアルタイムに単一ミトコンドリア機能の評価を可能にするライブアミナール中の無傷の組織のイメージング、共焦点走査顕微鏡法を用いた、新たなアプローチを記載する。まず、ミトコンドリアの対象とスーパーオキシドインジケータ、円順列黄色蛍光タンパク質（MT-cpYFP）を発現するトランスジェニックマウスを作製。麻酔をかけたMT-cpYFPマウスがカスタムメイドステージアダプタに固定され、タイムラプス画像は、Fをとっている後肢の露出骨格筋ROMをマウスはその後、犠牲にされ、心臓は37℃で、生理的なソリューションをランゲンドルフ灌流用に設定されている灌流心臓を共焦点顕微鏡ステージ上に特殊なチャンバ内に配置され、穏やかな圧力は、心臓を固定し、心拍誘導運動アーチファクトを抑制するために適用される。スーパーオキシド点滅毎秒1フレームの周波数でのリアルタイム二次元共焦点イメージングにより検出される。灌流溶液は、異なる呼吸基質または他の蛍光指示薬を含有するように修飾することができる。灌流はまた、虚血および再灌流などの疾患モデルを生成するように調整することができる。この技術は、無傷の組織およびインビボでの単一ミトコンドリアの機能を決定するためのユニークなアプローチである。

このプロトコルによると、単一のミトコンドリアのイベントのin vivoイメージングは、麻酔したマウスの骨格筋で行うことができますが灌流し、心臓内のその場での画像（ 図1） に続く。撮影条件の最適な設定は、そのまま筋肉組織の鮮明な画像を確保し、単一ミトコンドリアの解像度（ 図2）となります。 TMRMは、多くの場合、MT-cpYFPの場所を確?...

Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

共焦点走査顕微鏡は、生きた動物における灌流心臓または骨格筋​​におけるミトコンドリアの単一のイベントを画像化するために適用される。スーパーオキシドが点滅し、膜電位の変動などの単一ミトコンドリアのプロセスのリアルタイム監視は、生理学的に関連するコンテキストでかつ病理学的摂動の間、ミトコンドリア機能の評価が可能。

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング<em&gt;インビボ</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

confocal-imaging-single-mitochondrial-superoxide-flashes-intact-heart

Research

JoVE Journal

Biology

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

14.5K ビュー.  University of Washington. 共焦点走査顕微鏡は、生きた動物における灌流心臓または骨格筋​​におけるミトコンドリアの単一のイベントを画像化するために適用される。スーパーオキシドが点滅し、膜電位の変動などの単一ミトコンドリアのプロセスのリアルタイム監視は、生理学的に関連するコンテキストでかつ病理学的摂動の間、ミトコンドリア機能の評価が可能。 

ビデオ: Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

無傷の開発脳内の生体内の単一細胞のエレクトロポレーション

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

透過処理と無傷の細胞でミトコンドリア機能を評価するために高解像度呼吸計測

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

ショウジョウバエの調製と光コヒーレンス顕微鏡を用いてin vivoで心機能の縦イメージング（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング

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