私たちは、提示の仕事の重要な議論のための博士アリシアMattiazziに感謝します。この作品は、ALEにNIH（R01 HL-084487）からの助成金によってサポートされていました。

このプロトコルは、すべてのマウスの取り扱いは、UCマーセド施設内動物管理使用委員会（第2008から201）によって承認されました。インコを用いた実験は、科学研究のためのベネズエラ研究所（IVIC）の科学委員会によって確立された動物の使用のための一般的なポリシーに従って、1999年に実施しました。 1.ランゲンドルフセットアップの準備<ol><li> 140のNaCl、5.4のKCl、2のCaCl 2、1のMgCl 2、0.33 HPO 4、10グルコース、10 HEPES 2のNa：MMで、次の溶質濃度を含むタイロード溶液を調製します。 NaOHで7.4にタイロード溶液のpHを調整し、0.22μmのフィルターを通してソリューションをフィルタリングします。 </li><li> 60 mLの注射器と水平ランゲンドルフセットアップ11、 図4に示す12の全てのチューブにタイロード溶液をロードします。すべての気泡を排除することを確認します。 </li><li><stroNG&gt;図4に示すように沈めプラスチック「エアストーン」を使用して、100％のO 2と平衡タイロード液。 <ol><li> O 2タンクにプラスチックチューブを接続します。 </li><li> 60 mLシリンジ中のタイロード溶液に5 &quot;チューブを下げるためにプラスチック製のティーアダプタを追加します。 </li><li>タイロード溶液中に2意志バブルうちOので、5 &quot;チューブの端部にプラスチック製のエアストーンを取り付けます。 </li></ol></li><li>カニューレとして使用する針の周りの非吸収性縫合糸を配置します。針は、異なるソリューションでレトロ灌流を可能にするマニホールド（ 図4参照）に接続されています。最後に、心臓の大動脈は、針にカニューレされます。 </li></ol> 2.動物の準備と心解剖<ol><li>頚椎脱臼により安楽死の前に計量し、注入マウスをヘパリン（体重20グラムの例：マウス、20単位または200μLに注入する）15分。麻酔インコ応じトン頸椎脱臼を進め、あなたのIACUCプロトコルの動物使用ガイドoおよび。 注：8週齢のマウスまたは20グラムのインコを使用してください。 </li><li> 安楽死の後の胸腔から心臓を取り出します。マウスについて説明したように全く同じようにインコの心を抽出します。 <ol><li>エタノールでマウスの胸をきれいにしてください。 </li><li>解剖ハサミを使用して、下腹部に切開を行い、その後、首に向かって側面をカット。 </li><li>切断組織を引いて、それを突き止めます。 </li><li>振動板をカットします。心臓の損傷を防ぐために、振動板をカットするときは注意してください。 </li><li>肺と周囲の組織を削除します。 </li><li>それを圧迫することなく、心をすくうためにピンセットを使用してください。できるだけ長く大動脈をカットします。 </li></ol></li><li>約1 mLのタイロード溶液で小さな計量ボートに乗って心を運びます。 </li><li>非吸収性縫合糸を使用して、経由して水平ランゲンドルフ装置上に大動脈を結びます針。 2細かいピンセットの助けを借りて、心臓大動脈を結びます。タイロード溶液を含む60 mLの注射器と直列に位置バルブを開くことにより、レトロ灌流を開始します。 </li><li>心臓を10分間安定化させます。血液や染料をロードする前に心臓を取り囲む脂肪組織をきれいにするために、この時間を使用します。ピンセットで心の底部近くの脂肪組織を引き、小さな解剖ハサミを用いて切断。解剖顕微鏡の視野の下にこれを行うにしてください。 </li></ol> 3.細胞質ゾルのCa 2+測定：染料Rhod-2AMの準備<ol><li>染料の50μgのを含む染料メーカーが提供する特別なパッケージ化されたプラスチックバイアルにDMSO中の20％のプルロニック（非イオン性界面活性剤）の20μLを追加します。 </li><li>泡を避け、ピペッティングにより混和します。 </li><li>透明なガラスバイアルに特別にパッケージプラスチックバイアルから非イオン性界面活性剤と混合し、DMSOおよび色素を転送します。タイロードソリューの1 mLを加え透明なガラスバイアルにる。 </li><li>バス超音波処理器で15〜20分間超音波処理。 </li><li> 室温で30分間、蠕動ポンプを使用して染料を灌流。 <ol><li>染料チャンバ内で染料を置きます。 </li><li>マニホールドに接続された他の全てのチューブラインを圧縮する機械的クランプを使用してください。クランプは、マニホールドの上方に配置されています。これは、60 mLのシリンジに接続チューブ内に任意の逆流を防止します。 </li><li>染料を循環開始する灌流蠕動ポンプをオンにします。すぐに60 mLシリンジ、以下の3ウェイバルブを閉じます。 </li><li>位置心臓内に灌流された染料を再循環するために、心臓の隣に吸引蠕動ポンプに接続されている小さなチューブ。 </li></ol></li></ol> 4.イントラ-SRのCa 2+測定：染料マグ-Fluo4AMの準備<ol><li> Rhod-2AMと同様にマグFluo4AMを準備します。段階的な手順については、セクション3を参照してください。 </li><li> AFTE染料をロードR、レトロ灌流を開始するタイロード溶液を含む60 mLの注射器と直列に配置さ弁を開きます。マニホールドの上にクランプを削除してください。 </li><li>水平室にタイロード溶液を加え、37℃まで温めます。 </li><li>細胞質ゾル色素を除去するために45分間のタイロード液でレトロ灌流。 </li></ol> 5.膜電位測定：色素ジ8-ANEPPSの準備<ol><li>染料5mgを含有する染料バイアルに99％エタノールを5mLを加えます。 </li><li>リピーターピペットを用いて、500個々の1mLのプラスチックバイアルにアリコート10μLを。 </li><li> -20℃で高速真空や店舗で乾燥します。 </li><li>乾燥染料10μgのプラスチックバイアルにDMSO中の20％のプルロニックの20μLを追加します。 </li><li>泡を避け、ピペッティングにより混和します。 </li><li>プラスチックバイアルからメスシリンダー（10ミリリットル）にプルロニックおよび染料でDMSOを含むミックスを転送します。最終Voにタイロード液を追加5ミリリットルのLUME。 </li><li>バス超音波処理器で20〜25分間超音波処理。 </li><li>蠕動ポンプを用いて30分間心に灌流します。 3.5節で段階的な手順を参照してください。 </li></ol> 6。心外膜の信号を記録<ol><li>色素をロードした後、マニホールドの上にクランプを除去することにより、タイロード溶液で心をレトロ灌流。タイロード溶液を含む60 mLの注射器と直列に位置バルブを開きます。心を安定させるために10分間レトロ散らすタイロード溶液。 </li><li>タイロード溶液で水平室を記入し、37℃に浴温度をもたらすためにペルチェユニットの電源をオンにします。 </li><li> 心臓の表面に光ファイバを配置します。 <ol><li> 2 mLのピペット内部に光ファイバを配置し、マイクロマニピュレーターにピペットを取り付けます。 </li><li>少しLVの表面に対して光ファイバを押すようにマイクロマニピュレーターを使用してください。 </li></ol></li><li> 外部ょんのペース波動発生器によって制御される刺激で室温。 <ol><li>プログラム1ミリ秒の幅矩形パルスを提供するために、波発生器。 </li><li>外部で同期をとるための刺激を設定し、波発生器への外部入力に接続します。 </li><li>刺激の各出力に、最後に半田付け鍼灸針で線を接続します。 </li><li>約3mm離間心尖の両方で鍼を置きます。 </li><li>針が組織内に配置された後にのみ、感電を防止するために、刺激の出力をオンにします。 </li></ol></li><li>取得ソフトウェアでは、10 kHzに取得頻度を調整します。 </li></ol> 7 。心内膜信号を記録<ol><li>色素をロードした後、心臓を安定させるために6.1および6.2のステップを参照してください。 </li><li>光ファイバのサイズに関する鋭い23Gaポイントを使用して、中隔に近いLVに心臓の表面に小さな穴を作ります。 </li> &lt;LI&gt;マイクロマニピュレーターを用いて、心内膜に第一の光ファイバの位置決めを支援するために硝子体内手術強膜切開アダプタを置きます。 </li><li>外部から心臓のペース。 6.4ステップを参照してください。 </li><li>取得ソフトウェアでは、10 kHzに取得頻度を調整します。 </li></ol>

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本稿では、心筋細胞のex vivoでの機能を評価するために、ローカルフィールドの蛍光技術を説明するのに集中しています。接続された環境でのこれらの細胞の研究だけではなく、より生理的であるだけでなく、臓器レベルの病理を評価するための非常に適切です。興奮収縮連関（ECC）を基礎となる細胞事象は、細胞内Ca 2+動態（Rhod 2細胞質ゾルのCa 2+、マグFLUO4 SRのCa ...

心臓は心臓血管系の中心的な器官です。心臓の収縮は、細胞内の[Ca 2+] iの増加によって開始されます。細胞内Ca 2+放出における電気的興奮と変化の関係は、歴史的に、酵素的に解離した細胞1、2で研究されています。しかし、心臓の細胞は、電気的であり、代謝的及び機械的に結合された3、4。単離された場合、筋細胞だけでなく、物理的にカップリングしていないですが、異なる層から筋細胞を解離5中に混合されます。また、電圧クランプ条件下で単離された細胞の研究から浮上している巨大な利点、6、7、8電気シンシチウムALWAとして心の本質的な性質にもかかわらず、YS組織3に存在するものから細胞を解離する方法を機能的に異なるという問題を提起します。 本稿では、我々は、無傷の心臓における局所電場蛍光顕微鏡（LFFM）技術の使用により、心臓生理学の知識で得られた進歩について説明します。 LFFMは、細胞質ゾルのCa 2+、イントラ筋小胞体（SR）のCa 2+および膜電位などの複数の生理学的変数を測定するための蛍光指示薬を使用しています。これらの測定は、同時に心室圧9に関連して10、心電図9、電気的活動電位（APS）、イオン電流の記録とケージド化合物4、11のフラッシュ光分解を得ることができます。また、これらの測定値は、近い高い周波数で無傷の心臓をペーシングすることによって得ることができます生理率rと。いくつかの記事9、11、12、13、14は LFFM技術を使用して、我々のグループによって公開されているが、この技術に関連した技術的な複雑の推定は、心臓や他の臓器におけるex vivoの生理現象を研究する上での大規模な使用を妨げてきました。 LFFM技術（ 図1）は、組織と接触しているマルチモード光ファイバを使用して得られたエピ蛍光測定値に基づいています。任意の接触の蛍光イメージング技術と同様に、光学的分解能は、ファイバの直径と開口数（NA）に依存します。ファイバのNAが高い小径の測定の空間分解能を増加させます。 NASおよび繊維径は、0.22から0.66まで、50ミクロンからそれぞれ1mmとの範囲とすることができます。にNAのしわは、より大きな立体角から到来する光子を受け入れることによって信号対雑音比（S / N）に信号を改善します。落射蛍光装置として機能させるために、光ビームは、非球面レンズまたはレンズとファイバ一致NA落射蛍光対物レンズで光ファイバに集光されます。このマッチングは、励起用およびフルオロフォアによって放出された光子をバック収集するためのエネルギー伝達を最大化します。 組織内にロードされた外因性の蛍光標識を励起するために、異なる光源及び照明モードを利用することができます。パルス局所視野蛍光顕微鏡3、12（PLFFM）を使用して我々の先駆的な研究では、低コストのピコ秒レーザー（ 図1a、PLFFM）を用います。光源のこのタイプは、実質的に起因する色素を漂白することなく、照明領域の下のフルオロフォア分子の励磁大きな画分の大きな利点を有しています短パルス持続時間12へ。また、超短パルスの使用は、色素12の蛍光寿命の評価を可能にしました。蛍光寿命は、Ca 2+に結合した色素分子の割合を定量化するために使用することができる特性です。残念なことに、パルス間の振幅のパルスと変動の時間的なジッタは、色素に結合するリガンドによって生じる蛍光の変化が大きい場合に、この実験ストラテジーの適用を制限します。 連続波（CW）レーザは、通常LFFM（ 図1b、CLFFM）の主照明源として使用されます。レーザビームは、連続的に、組織を照明することができるか、ferroelectrically調節することができます。ビームの強誘電体の変調は、光のマイクロ秒のパルスの生成を可能にします。この変調は、外部ハードウェアによって制御することができます。この手順では、劇的トンを低減するだけでなく彼の光パルスの時間的ジッタだけでなく、異なる波長の光を混合することができます。光の混合は、異なるレーザからの多重線によって行われます。結果として、異なるスペクトル特性を有する複数の色素は、例えば、生理学的変数の種々の測定を実行するために励起することができるRhod-2細胞質ゾルの Ca 2+のための、MagFluo4イントラSR の Ca 2+、ジ-8-ANEPPS用用膜電位。 LFFM光源としてレーザは、存在する種々の利点があるが、他のタイプの光源は、発光ダイオード（LED）などを用いることができます。この場合、励起光源は、InGaNのLED（ 図1c、PLEDFM）から成っていました。伝導帯から電子が価電子帯の正孔と再結合するときのLEDにおいて、光子が自然に放出されます。固体レーザとの違いは、発光が他の光子によって刺激されないことです。これは、非コヒーレントなビームをもたらすとLEDの広いスペクトル放射。 ハイパワーLEDの異なる種類を使用することができます。 Fluo-4またはマグのFluo-4を使用して記録ディ-8-ANEPPSを使用して、APの記録のためにおよびCa 2+過渡現象のために、我々は485nmの（青）で、典型的なピーク発光および20nmの半分の幅を有するLEDを使用しました（ 図1D）。 Rhod-2で記録した Ca 2+過渡現象のために、LEDは540nmの（緑）、35 nmの（ 図1D）の半分の幅で、典型的なピーク発光を有していました。 LEDは、バンドの波長で発光するので、それらのスペクトル放射を絞り込むためのフィルタを必要とします。また、パルス光は、20マイクロ秒の持続時間で1.6キロヘルツのレートで生成することができます。 LEDは、高速パワーMOSFET電界効果トランジスタを用いてパルスしました。異なる指標との同時記録は時間多重LEDによって行うことができます。残念ながら、LEDによって放射される光は、レーザ光に比べて光ファイバに集中することはより困難です。このように、私たちの主な欠点LEDをることは、それらの放出プロファイルは、メイン軸からの角度変位（±15°）を持っており、補助光学部品は、それを修正するために使用しなければならないことです。 前述の光学的構成のすべてにおいて、励起光は、ダイクロイックミラーを用いて反射されます。ビームは、その後、組織に配置されているマルチモード光ファイバに非球面レンズと顕微鏡対物レンズによって集束されます。任意の落射蛍光装置のように、ダイクロイックミラーは、また、放射された光から励起を分離するのに役立ちます。放出された光スペクトルは、任意の反射された励起を除去するために、バリアフィルタを通って戻って移動します。最後に、放出された光は光検出器（ 図1）上に目的に集束されます。 光から電流への変換は、シリコンアバランシェフォトダイオードによって実行されます。これらのダイオードは、高速応答、低光の検出を可能にする高感度を持っています。ザアバランシェフォトダイオードによって生成される光電流は、2つの方法で増幅することができる：トランスインピーダンス増幅器は、帰還抵抗素子（ 図1eの）を有するまたは電圧（ 図1F）に電流を変換する積分器。第一のアプローチを使用して、出力電圧は、光電流と帰還抵抗に比例します。ピコ秒レーザパルスの抵抗検出の典型的な例は、 図2a、2b及び2cに示されています。パネル2aは、トランスインピーダンスアンプの出力を示し、パネル2bは、アスタリスク（*）で示された区間の時間展開を示しています。ピーク追跡アルゴリズムは、蛍光応答12のピーク（赤）とベース（緑）を検出するために実装されました。ベース蛍光の測定は、周囲LIGによって導入アバランシェフォトダイオードの暗電流および干渉の両方の情報を提供しますHTと電磁結合。ピークと塩基の表現は、 図2cに示されています。この図は、鼓動しているインコの心臓の心周期中の Ca 2+に結合した色素（Rhod-2）が発する蛍光を示しています。 第二の方法では、積分器の出力電圧は、電流と容量性フィードバック（ 図2D、2E、及び2F）の関数です。 図2Fは、2つの連続した統合サイクルを示していますなし外部照明およびパルスLEDから照射された光パルスと第二と第一。詳細な説明は 、 図2Gおよび2Hに示されています。このアプローチは、より面倒が、によりフィードバックコンデンサにおける熱雑音が存在しないため、より大きなS / Nを提供します。機器は、励起光の全ての制御および多重化を生成し、ヘッドステージの統合及び解像度を指示するタイミング段階を含みますらの期間。これは、通常、データのオンライン回帰を計算することによって、統合された出力信号のデジタル分化を行うデジタル信号処理回路を用いて行われます。抵抗性フィードバックを用いた場合には、任意のA / D取得ボードを使用することができます。 最後に、我々のLFFM技術は、非常に汎用性であり、複数の領域から記録するように適合させることができます。光路中にビームスプリッタを追加すると、私たちは2本の光ファイバに光を分割することができます。各光ファイバは、その後、外因性の蛍光プローブから、独立して、励起する組織と記録放出の異なる領域に配置することができます。この変更は、生理学的変数にどのように影響するかを解剖学的な地域差を評価するために私達を許可します。 図3は、2本の光ファイバを経電気または細胞内の[Ca 2+] iレベルのウィットを測定するために使用されるようにCW励起光を分割するために使用されるビームスプリッタを示します時間マイナー侵襲。経た信号は、1つの心内膜上のファイバと心室壁の心外膜層の上に他方を配置することによって記録することができます。したがって、LFFM技術は、異なる領域における細胞シグナルの経時変化を測定する能力を有しており、地域の変化は、病理学的状況で起こるかどうかをテストするために使用することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Sigma</td>
			<td>10049-21-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride solution</td>
			<td>Sigma</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution</td>
			<td>Sigma</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hyrdoxide</td>
			<td>Sigma</td>
			<td>S-8045</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2um nylon membrane filter</td>
			<td>Whatman</td>
			<td>7402-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold MPP</td>
			<td>Warner</td>
			<td>64-0216&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>21G1.5 Precision glide needle</td>
			<td>B-D</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Braided silk string (non-absorbable surgical suture)</td>
			<td>AllMech Tech</td>
			<td>LOOK-SP105</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium injection 1000USP/mL</td>
			<td>AllMech Tech</td>
			<td>NDC63323-540-11</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO D8779</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma</td>
			<td>856925-71-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 20% solution</td>
			<td>Biotium</td>
			<td>59004</td>
			<td></td>
		</tr>
		<tr>
			<td>Materflex C/L peristaltic pump</td>
			<td>Cole-Parmer</td>
			<td>77122-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Isostim stimulator</td>
			<td>World Precision Instruments</td>
			<td>A320RC</td>
			<td></td>
		</tr>
		<tr>
			<td>Waveform generator</td>
			<td>Teledyne Lecroy</td>
			<td>WaveStation 2012</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner FS20</td>
			<td>Fisher Scientific</td>
			<td>1533530</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing ID:1/32&#34; OD:3/32&#34; Wall 1/32&#34;</td>
			<td>Component Supply</td>
			<td>TET-031A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing ID:3/32&#34; OD:5/32&#34; Wall 1/32&#34;</td>
			<td>Component Supply</td>
			<td>TET-094A</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter luer lock to 3-way valve</td>
			<td>Cole-Parmer</td>
			<td>EW-31200-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Tee adapters and plastic fittings</td>
			<td>Cole-Parmer</td>
			<td>6365-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic clamp</td>
			<td>WaterZoo</td>
			<td>2465</td>
			<td></td>
		</tr>
		<tr>
			<td>Peltier</td>
			<td>TE Technology</td>
			<td>TE-127-2.0-2.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhod-2AM</td>
			<td>ThermoFisher Scientific</td>
			<td>R1245MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-8-ANEPPS</td>
			<td>ThermoFisher Scientific</td>
			<td>D3167</td>
			<td></td>
		</tr>
		<tr>
			<td>Mag-Fluo-4AM</td>
			<td>ThermoFisher Scientific</td>
			<td>M14206</td>
			<td></td>
		</tr>
		<tr>
			<td>Acupunture needles</td>
			<td>LHASA</td>
			<td>TC1.20x13</td>
			<td></td>
		</tr>
		<tr>
			<td>60mL BD syringe with luer-lok</td>
			<td>Fisher Scientific</td>
			<td>14-820-11</td>
			<td></td>
		</tr>
		<tr>
			<td>LabView</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Speed vacuum Eppendorf Vagufuge</td>
			<td>Fisher Scientific</td>
			<td>07-748-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital signal processing circuit DSP TMS 320</td>
			<td>Texas Instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Longpass Dichroic mirror 567nm</td>
			<td>ThorLabs</td>
			<td>DMLP567L</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective 10X NA 0.25 DIN AchromaticFinite Intl Standard Objective</td>
			<td>Edmund Optics</td>
			<td>Stock# 33-437</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective 20X NA 0.40 DIN Achromatic Finite Intl Standard Objective</td>
			<td>Edmund Optics</td>
			<td>Stock# 33-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Longpass colored glass filter 590 nm&#160;</td>
			<td>ThorLabs</td>
			<td>FGL590</td>
			<td></td>
		</tr>
		<tr>
			<td>Green Nd-YAG laser 532nm, 500mW</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator for laser</td>
			<td>Siskiyou</td>
			<td>MX130R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode fiber optic 200&#181;m NA 0.39</td>
			<td>ThorLabs</td>
			<td>FT200UMT</td>
			<td></td>
		</tr>
		<tr>
			<td>LEDs blue</td>
			<td>Lumileds</td>
			<td colspan="2">L135-B475003500000</td>
		</tr>
		<tr>
			<td>LEDs green</td>
			<td>Lumileds</td>
			<td colspan="2">L135-G525003500000</td>
		</tr>
		<tr>
			<td>Cube beam splitter, non-polarizing</td>
			<td>ThorLabs</td>
			<td>BS007</td>
			<td></td>
		</tr>
		<tr>
			<td>36&#34; Length, Dovetail Optical Rail</td>
			<td>Edmund Optics</td>
			<td>54-402</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Width, Dovetail Carrier</td>
			<td>Edmund Optics</td>
			<td>54-404</td>
			<td></td>
		</tr>
		<tr>
			<td>0.75&#34; Travel, micrometer stage</td>
			<td>Edmund Optics</td>
			<td>37-983</td>
			<td></td>
		</tr>
		<tr>
			<td>Dovetail optical rail 3&#34;</td>
			<td>ThorLabs</td>
			<td>RLA075/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dovetail rail carrier 1&#34;</td>
			<td>ThorLabs</td>
			<td>RC1</td>
			<td></td>
		</tr>
		<tr>
			<td>Avalanche photodiode Helix 902</td>
			<td>Digi-Key</td>
			<td>HELIX-902-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective holder XY Translator&#160;</td>
			<td>ThorLabs</td>
			<td>ST1XY-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum breadboard 6&#34;x6&#34;</td>
			<td>ThorLabs</td>
			<td>MB6</td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus otpical table 4&#39;x6&#39;</td>
			<td>ThorLabs</td>
			<td>T46HK</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel cap screws</td>
			<td>ThorLabs</td>
			<td>HW-KIT5/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic&#160; sheet</td>
			<td>Home Depot/Lowes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard Silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy gel&#160;</td>
			<td>Walmart/Home Depot</td>
			<td>2-part 5 min clr 1oz</td>
			<td></td>
		</tr>
		<tr>
			<td>21G1.5 Precision glide needle</td>
			<td>B-D</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>23G Precision glide needle</td>
			<td>B-D</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting base, 25mmx75mmx10mm</td>
			<td>ThorLabs</td>
			<td>BA1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire shelve posts 36&#34;</td>
			<td>Alera</td>
			<td>AALESW59PO36SR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire shelves</td>
			<td>Alera</td>
			<td>ALESW582424SR</td>
			<td></td>
		</tr>
		<tr>
			<td>Post and angle clamp</td>
			<td>ThorLabs</td>
			<td>SWC/M-P5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe for dye chamber</td>
			<td>Wheaton</td>
			<td>W851020</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber stopper</td>
			<td>Home Science Tools</td>
			<td>CE-STOP01C</td>
			<td></td>
		</tr>
	</tbody>
</table>

local field fluorescence microscopy: imaging cellular signals in intact hearts

心臓では、分子シグナリング研究は、通常、単離された筋細胞で行われています。しかしながら、このような虚血および不整脈などの多くの病的状態は、完全に全臓器レベルで理解することができます。ここでは、無傷の心の中の細胞の信号の測定を可能にするローカルフィールド蛍光顕微鏡（LFFM）の分光技術を提示します。技術は、蛍光シグナルを記録するランゲンドルフ灌流心臓および光ファイバの組み合わせに基づいています。 LFFMは正常および病的条件下で心を勉強する心血管生理学の分野で様々な用途があります。複数の心臓の変数は異なる蛍光指示薬を用いてモニターすることができます。これらは、細胞質ゾルの[Ca 2+]、イントラ筋小胞体の[Ca 2+]および膜電位が含まれます。外因性蛍光プローブが励起され、放出される蛍光はLFFM落射蛍光技術の三つの異なるアレンジで検出します本論文での。これらの技術の中で中心的な違いは、励起用の励起光が変調されている方法で使用される光源の種類です。連続波LFFM（CLFFM）は、励起用の連続レーザ光を使用しながら、パルスLFFM（PLFFM）は、レーザ光パルスを使用しています。最後に、発光ダイオード（LED）は、第3の光源として使用しました。この非コヒーレント構成は、パルス状のLED蛍光顕微鏡（PLEDFM）と呼ばれています。

心内膜および心外膜における APおよびCa 2+ 過渡現象 心室壁を横切って信号を比較するためには、光ファイバは、心外膜での心内膜及び他方に配置されています。心外膜からのものと心内膜から記録されたAPの形態を比較すると、貫機能を評価するための最良の方法?...

Watch this Scientific Journal Video about Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts at JoVE.com

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

心臓では、分子事象は器官の電気的および収縮機能を調整します。ここで紹介する地元の視野蛍光顕微鏡技術のセットは、無傷の心の中の細胞の変数の記録を可能にします。心機能を定義するメカニズムを識別することは心が病的な状況下でどのように機能するかを理解する上で重要です。

ローカルフィールド蛍光顕微鏡：無傷の心の中に携帯電話の信号のイメージング

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

local-field-fluorescence-microscopy-imaging-cellular-signals-intact

Research

JoVE Journal

Biology

8.2K ビュー.  University of California, Merced. 心臓では、分子事象は器官の電気的および収縮機能を調整します。ここで紹介する地元の視野蛍光顕微鏡技術のセットは、無傷の心の中の細胞の変数の記録を可能にします。心機能を定義するメカニズムを識別することは心が病的な状況下でどのように機能するかを理解する上で重要です。 

ビデオ: Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

広視野顕微鏡を用いて長期的なイメージング哺乳類細胞

生物学

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

全反射顕微鏡による網膜双極細胞におけるイメージングのエキソサイトーシス

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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 ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

迅速な分析および蛍光顕微鏡画像の探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

タンデム高圧凍結および透過型電子顕微鏡のための植物組織の急速凍結置換

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

ハイコンテンツ顕微鏡を用いた細胞レドックスプロファイリング

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

3 D 蛍光顕微鏡を用いたウイルス転写工場の機能イメージング

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...