私たちは、優れた技術サポートのためにダニエル・ブカラ、ナタリー・アルトメイヤーとFundaケイに感謝します。私たちは、原稿を編集するためのジョナサン・コットン、グレッグ・ボーデンとポールSoubiranに感謝します。この作品は、CRC 156（プロジェクトC3）を通じてヴェルナーシーメンス財団とエバーハード・カールズ大学テュービンゲン（「」Promotionskolleg「」）の医学部でとDFGによってサポートされていました。

本書に記載されているすべての手順は、実験動物のケアと使用のガイドラインと国際基準に従い、ドイツのテュービンゲン国家委員会の動物福祉倫理委員会によって承認されました。 8〜12週齢のBALB / cおよびC57BL / 6マウスを12時間：12時間明/暗サイクルに保ち、IVCおよび22±1℃の標準環境条件で2〜5の群で水および食べ物は自由にアクセスできます 。 材料準備<ol><li>注射直前にそれぞれのデータシートに従って近赤外蛍光イメージングのためのOI色素を希釈する。 インビボで MMPを測定するための活性化可能なOI色素（680nmでの励起を伴う市販のプローブ）は、1.5mLの1×PBS中で20nmolの濃度で使用する準備ができている。使用する前に、溶液を静かに振り混ぜるかボルテックスする。 OI染料は、光から保護されている場合、最大6ヶ月間2〜8℃で保存することができます。 </li&gt; <li> 静脈内 （IV）注射は、静脈カテーテルを準備します。 0.9％生理食塩水で満たされた20 U（0.5 mL）をインスリンシリンジ（50mLのヘパリンの10個の噴射単位を含有する）、およびポリエチレンカテーテルに取り付けられた30ゲージの針を使用します。 OI染料のマウスあたり2 nmolのの推奨用量を注入します。 </li></ol>関節リウマチや慢性接触過敏反応の2誘導<ol><li> 関節リウマチ： <ol><li> RAを誘導するために1×PBS（200μL）で1：グルコース-6-リン酸イソメラーゼ（GPI）1に対する抗体（AB）を含む（K / BXNマウス10から得られた）血清の100μLに希釈します。 -80°Cで希釈した血清を保管してください。 RAの誘導のための詳細な手順はMonach らによって記載されています。 8。 </li><li> RAを誘導するために、その尾により穏やかに各マウスを持ち上げ、Tの0日目に希釈した血清を腹腔内（IP）の200μLを注入彼は実験します。注射後、マウスをそのケージに直接戻す。 </li><li>オプションで、AB注射の前に各足首の足首の直径を測定し、「関節炎スコア」を定義する（ 図1A ）。機械的測定装置（マイクロメーター）を用いてGPI血清後6日目まで毎日足首腫脹の測定を継続する。 </li><li> RAマウスの尾静脈に生体内 MMP活性を測定するために、活性化可能なOI色素を注入する（ステップ1.1~1.2） GPI血清注射後5日目およびコントロールマウスの尾静脈に注射した。尾静脈注射の24時間後に光学イメージング実験を行う。 </li></ol></li><li> 接触過敏反応： <ol><li> C57BL / 6マウスの感作のために、アセトン/オイルの4：1混合物に溶解した5％TNCB溶液を調製する。 </li><li> 100％酸素（1.5L /分）で気化した1.5％イソフルランを用いてC57BL / 6マウスを麻酔する。マウスを麻酔したら、それを自己狂犬病<html> （1.5体積％のイソフルランチューブに接続した切断5mLのポリエチレンシリンジ）で麻酔を維持する。麻酔をかけた動物に獣医用軟膏を塗布し、麻酔下で眼の乾燥を防ぎます。 </li><li>小さな動物の毛のトリマーを使用して慎重に（2×2 cm）腹部を剃る。動物の中で非特異的な蛍光シグナルを引き起こし、研究結果に影響を与える可能性があるため、皮膚を傷つけないでください。 </li><li>動物を感作するために100μLピペットを使用して、剃毛した腹部領域で5％TNCB溶液80μLをゆっくりと適用する。角膜の損傷を避けるために、マウスの目が寝具に接触しないように注意しながら、ケージに軽く戻してください。 </li><li>感作の6日後に、1％TNCB溶液（アセトン/オイルの9：1混合物に溶解）を調製し、ピペットを用いて右耳に20μLをかけて急性および慢性CHSRを誘発する。「&gt; 1 <ol><li> 100μLピペットを用いて、1％TNCB溶液20μLと右耳の両側に第1チャレンジで始まり、CHR（ 図1B）を引き出すために5回（感作後15日目）に毎秒日チャレンジを繰り返します。マイクロメータ測定装置を使用して、毎日の耳の厚さを測定します。 </li></ol></li><li> CHRマウスにおいてチャレンジ後12時間のin vivoでの MMPの活性を測定する活性化OI色素（680nmにおける励起を有する市販のプローブ）（ステップ1.1から1.2）を注入します。光学イメージング実験の24時間後に尾静脈注射を行う（IV）。 </li></ol></li></ol>光イメージングのための3動物の準備<ol><li>スイッチマウス固形飼料に使用OI剤の蛍光信号の低または非蛍光飼料（ 例えば 、遊離マンガン）（700nmの周りの）自家蛍光の干渉を回避するために（少なくとも3日間撮影前）。 </li><li>で動物を置きます酸素（1.5L /分）で気化した1.5vol％イソフルラン（酸素は空気で置換することができるが、1回の実験で標準化する必要がある）を用いて麻酔する。 </li><li>マウスが目に見えて麻酔されたら、それを自作ノーズコーン（1.5体積％イソフルランチューブに接続したカット5mLポリエチレンシリンジで構築）に置き、麻酔を維持する。 </li><li>小動物ヘアトリマーを使用して、動物を注意深く標的部位（2cm×2cm）に剃ります。動物の中で非特異的な蛍光シグナルを引き起こし、研究結果に影響を与える可能性があるため、皮膚を傷つけないでください。 注：HIRは、関心領域（ROI）に依存して、OI中に蛍光シグナルを吸収することができる（マウス株に依存し、多かれ少なかれ吸収が観察される）。 </li><li> 静脈内注射のために、暖かい水の中にマウスの尾を置き、血管拡張を誘発し、アルコールで注射部位の皮膚を静かに浄化し、カテーテルを尾の先端サイト。尾静脈に20°の角度で、針の端を「カット」と注射器を再懸濁することによってカテーテルの正確な配置をテスト置きます。 </li><li>カテーテルが正しく配置されている場合は、シリンジを交換し、OIのプローブ（2ナノモル）を注入します。注入後、シリンジを交換し、完全にポリエチレンチューブのデッドボリュームをクリアするために、0.9％の食塩水の25μLを注入。 注： インビボで MMP活性を測定するために使用OI色素（680nmにおける励起を有する市販のプローブ）の組織半減期は72時間です。完全なクリアランスを確保するために、色素の再注入は、以前の7日前に注射後よりも推奨されていません。 </li></ol> 4.光イメージング<ol><li>視野（FOV）の中心でOI-スキャナのブラックボックスに黒いプラスチックまたは紙シートを置きます。 </li><li>測定プロトコルを設定し、右の波長を選択し（励起：680±10 nmおよび電子ミッション：700±10 nm）を、撮像パラメータ。 注：一部のOIシステムでは、複数の画像形成色素の設定が事前に定義されています。 </li><li>蛍光イメージングのために正しいプロトコルを選択するために、（製造業者によって提供された）画像ソフトウェアを開き、システムを初期化します。ほとんどのCCDカメラは、その使用温度まで冷却する必要があり、これは10分かかることがあります。システムの準備ができるまで、信頼性の高い結果を得るために、待ちます。 </li><li> in vivoイメージングシステム取得制御パネルがポップアップ観察し、選択された各フィルタ対は、配列中の1枚の画像を表します。この場合には、を有する市販の染料及びそれぞれ680±10nmから700±10nmでの励起および発光波長のフィルタのペアで一つの画像を取得し、（押し「を取得配列」）の測定を開始します。より詳細な手順については、製造元のマニュアルを参照してください。 </li><li>適切な画像にラベルを付け、「イメージの編集に情報を保存します「ラベル取得」ウインドウが表示され、「取得シーケンス」の後にポップアップ表示されます。 </li><li> OI色素を注射する前に各動物のベースラインスキャンを行うか、またはナイーブコントロール動物を用いてバックグラウンドシグナルを区別する。 </li><li> OI色素の尾静脈注射後、OIシステムにおいて最も高いシグナルを測定する位置で、動物をFOVの中心に置き、測定を開始する。 注記：重要：低体温はイメージング剤の分布に大きな影響を与える可能性があります。動物の低体温を避けるために、ステージが37℃まで加熱されていることを確認してください。イメージングは​​、1〜5匹のマウスの群の動物と同時に行うことができる。同時に測定する動物の数に応じて、FOVのサイズを選択します。各マウスが見えるかどうかを確認するには、明るいフィールド画像を撮ることができます。 </li></ol> 5.データ解析注：次の画像ソフトウェアを使用してデータ分析を実行します製造業者のプロトコル。 <ol><li> RAの測定には、 図2に示すように校正された光子放射の単位を使用しますが、慢性CHSRは信号強度として％（効率）で描かれます。 </li><li> ROIを手動で描画するには、ツールプレートを使用します。 RA画像の分析のために、標準化された円を使用し、各動物のすべての足首および足の中で最も高いシグナルの周りに配置する。 CHSRを分析するには、明視野画像に従って左右の耳全体にROIを配置します。 </li><li>描かれた特定のROIの光子放出またはシグナル強度値を測定するには、「測定」を押します。システムは、描写的な統計解析のために描かれたROIの値を提供する。 </li></ol>

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S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+targeting+of+MMP-2/9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation).">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li><li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques.">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li><li>Wagner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET:+radiosynthesis+and+initial+small-animal+PET+studies.">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li><li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe.">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li><li>Vogl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alarmin+S100A8/S100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity.">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593 (2014).</li></ol>

著者は何も開示することはありません。

OIは、前臨床研究インビボ分子イメージングにおける非侵襲性のために非常に有用な迅速かつ安価なツールです。 OIの特定の強さは、炎症応答のような非常に動的なプロセスを監視する能力です。また、OIは、1つの数日から数週間の範囲の、長期間にわたって病気の経過を追跡することを可能にします。 それは非常に時間とコスト効率的であるようにOIは、...

関節リウマチ（RA）または尋常性乾癬などの自己免疫疾患は、遅延型過敏症反応（DTHR）として分類される。 RAは、びらん性滑膜炎および関節破壊によって特徴付けられる一般的な自己免疫疾患である。炎症細胞の浸潤と増殖、炎症細胞の発現の増加がパンヌス形成、軟骨および骨破壊をもたらすことを実証している2。マトリックスメタロプロテイナーゼ（MMPs）によるコラーゲンなどの細胞外マトリックス分子の切断は、組織の変換や血管新生に不可欠であり、組織の破壊を引き起こす。 5、6接触過敏反応（CHR）は、酸化的バーストをもたらす好中球の凝集によって特徴付けられる。 RAと同様に、CHRのMMPはインボルブ慢性炎症を確立するために、組織変換、細胞移動および血管新生に供される。 RAを調べるために、グルコース-6-リン酸イソメラーゼ（GPI） - 血清注入マウスモデルを使用した。 GPIに対する抗体を含有するトランスジェニックK / BxNマウスの血清をナイーブBALB / cマウスに注射した後、GPI血清注入後6日目に最大24時間以内にリウマチ性炎症が発生し始めた（1.1参照）。慢性CHRを分析するために、C57BL / 6マウスを腹部のトリニトロクロロベンゼン（TNCB）で感作した。右耳に感作後1週間目から5回までチャレンジした（1.1および1.2も参照）。 非侵襲的小動物OIは、前臨床研究で主に使用されている蛍光、化学発光および生物発光シグナルの生体内調査に基づく技術である。得られた半定量的データは、分子健康な器官および組織におけるウラル機構ならびに罹患実験動物モデル、及び縦フォローアップ測定値（ 例えば 、インビボでの治療的応答プロファイルを評価するため）を可能にします。縦断的研究の大きな利点は、同じ動物ではなく、時点ごとに異なるマウスを用いて、いくつかの時点でフォローアップ試験で測定することができるように、動物の数が減少することです。 OIの解像度は、臓器の詳細な機能イメージングおよび実験動物におけるさらに小さな組織構造を可能にします。 狭い透過スペクトルを持つ特定の励起および発光フィルタの使用、遮光「暗箱」と-70℃に多くの装置で冷却された敏感な電荷結合素子（CCD）カメラ、によって散乱された光に対する保護、蛍光シグナルの高度に特異的かつ高感度な測定が可能となります。 excitation-と蛍光剤を使用し、近赤外蛍光ウィンドウの発光スペクトル（650から950 nm）は、信号対雑音比を大幅に向上させることができます。近赤外蛍光ウィンドウはヘモグロビン及び水による信号の比較的低い吸収ならびに低バックグラウンド自己蛍光によって特徴付けられます。 9これは、小動物の組織で最大2センチ侵入深さを可能にします。 OI-プローブが直接（蛍光標識抗体による）目標に対処することができるか、（プロテアーゼにより）標的組織中で活性化することができます。活性化OIプローブを伴う別のドメインへの分子内励起エネルギーを伝達消光部分、に蛍光共鳴エネルギー移動（FRET）にそれらの不活性化形態の光学的にサイレントです。染料は（例えばプロテアーゼによって）切断された場合にエネルギーはもはや分子内に移し、蛍光シグナルがOIによって検出することができるではありません。これは、高specificitとOIプローブの設計を可能に明確な生物学的プロセスと優れた信号対雑音比が得られます。 以下のプロトコルは、動物の調製、in vivoでの MMP-2、-3、-9および-13活性および炎症の2つの実験モデル（RA、CHR）を画像化するための活性化可能OIプローブを用いたOI測定を詳細に説明する。

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			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
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		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
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			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
		</tr>
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			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
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			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
		</tr>
		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
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		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
		</tr>
		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
		</tr>
		<tr>
			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
		</tr>
		<tr>
			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
		</tr>
		<tr>
			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
		</tr>
		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

この論文では、炎症の2つの異なるマウスモデル：慢性関節リウマチ（RA）および接触（慢性関節リウマチ） におけるin vivo蛍光イメージング（OI） を介して 、活性化可能な蛍光プローブによるマトリックスメタロプロテイナーゼ（MMP）活性を画像化するための非侵襲的方法を記載する。過敏反応（CHR）モデル。近赤外（NIR）ウィンドウ（650〜950nm）の波長を持つ光は、650nm未満の波長に比べて、より深い組織浸透と最小限の信号吸収を可能にします。蛍光OIを用いることの主な利点は、異なる動物モデルで安価で、迅速かつ容易に実施できることである。 活性化可能な蛍光プローブは、それらの不活性化状態では光学的にサイレントであるが、プロテアーゼによって活性化されると高度に蛍光性になる。活性化されたMMPは、組織破壊をもたらし、遅延型過敏症反応（DTHR）（例えば、RAおよびCHR）における疾患の進行にとって重要な役割を果たす。さらに、MMPは、キー軟骨および骨の分解のためのプロテアーゼおよび炎症性サイトカインに応答して、マクロファージ、線維芽細胞や軟骨細胞によって誘導されます。ここでは、MMP-2のようなキーのMMPによって活性化されたプローブ、-3、-9および-13を使用して、同様に6日間の疾患誘導後RAおよび対照マウスにおけるMMP活性の近赤外蛍光OIのための撮影プロトコルを記述する急性（1Xチャレンジ）と健康の耳に比べ右耳の慢性（5倍の挑戦）CHRを持つマウスのように。

ナイーブBALB / cマウスにおいて関節リウマチ（RA）を誘発するために、0日目にGPIに対する自己抗体（1×PBSで1：1希釈）を腹腔内注射した。このGPI血清における最大炎症（足首腫脹） RAモデルは注入11日後に投与される。したがって、2nmolの活性化可能なOI色素を調製し、5日目に関節炎マウスおよび健常対照動物の尾静脈に静脈注射した。注射の24...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

この論文は、炎症の二つの異なる実験モデルにおいて、キーマトリックスメタロプロテアーゼのインビボ活性を視覚化するために活性化光イメージングプローブを用いた蛍光イメージングの応用について説明します。

非侵襲的<em&gt;インビボ</em&gt;活性化可能な蛍光イメージング剤を用いた炎症性MMP活性の蛍光イメージング

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Research

JoVE Journal

Immunology and Infection

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

8.7K ビュー.  Eberhard Karls University of Tübingen. この論文は、炎症の二つの異なる実験モデルにおいて、キーマトリックスメタロプロテアーゼのインビボ活性を視覚化するために活性化光イメージングプローブを用いた蛍光イメージングの応用について説明します。 

ビデオ: Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

動的な生細胞イメージングのための宿主 - 病原体相互作用の研究のための光トラップの使用

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

異なる動物モデルにおけるNADPHオキシダーゼ活性の生物発光イメージング

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

ゼブラフィッシュ幼生における播種性カンジダ症の非侵襲的イメージング

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物発光細菌のイメージング<emインビボで&gt;</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

マウスのリンパ脈管構造の非侵襲的光イメージング

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

急性及び慢性炎症を区別するために生体内イメージング法における

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

マウスリンパ球標識による<sup&gt; 64</sup以下のためのターゲット設定&gt;のCu-抗体の受容体<em&gt;インビボ</em&gt; PET / CTによる細胞輸送

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

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白血球ホーミングと移行の非侵襲的イメージング in vivoで

動的な生細胞イメージングのための宿主 - 病原体相互作用の研究のための光トラップの使用

異なる動物モデルにおけるNADPHオキシダーゼ活性の生物発光イメージング

ゼブラフィッシュ幼生における播種性カンジダ症の非侵襲的イメージング

生物発光細菌のイメージング

マウスのリンパ脈管構造の非侵襲的光イメージング

急性及び慢性炎症を区別するために生体内イメージング法における

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング

マウスリンパ球標識による

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出