この作品は、寛大に神経疾患・脳卒中（NIH / NINDS）助成金1R01NS086981とコンラッドN.ヒルトン財団の健康/国立研究所の国立研究所によってサポートされていました。我々は、共焦点顕微鏡との貴重な援助のために博士ローランBentolila博士マシューSchiblerに感謝します。私たちは、CLARITYフォーラム（http://forum.claritytechniques.org）に寄与しているものに感謝します。我々は、特にこの魅力的な技術を教えるために、彼の研究室を開放するための博士カールDeisserothに感謝します。著者らは、脳マッピング医学研究機構、脳マッピング支援財団、ピアソン・ラブレス財団、アーマンソン財団、キャピタル・グループ会社慈善財団、ウィリアムM.とリンダR. Dietel慈善基金、およびノー​​ススター基金からの寛大な支援に感謝してい。この刊行物で報告された研究はまた、部分的に研究資源のための国立センターと国家のディレクターのオフィスでサポートされていました受賞番号C06RR012169、C06RR015431、およびS10OD011939下の健康のアル研究所。内容はもっぱら著者の責任であり、必ずしも国立衛生研究所の公式見解を示すものではありません。

すべての実験は、施設内動物管理使用委員会（IACUC）のガイドラインに従って行われました。 THY1-YFP、PLP-EGFPおよびPV-TdTomatoマウスをこれらの実験に使用したが、蛍光タンパク質を発現している任意のマウスは正常にクリアして画像化することができます。 1.組織標本注意：パラホルムアルデヒド（PFA）とアクリルアミドが毒性と刺激物です。ドラフト内で、適切な個人用保護具（白衣、手袋、目の保護）で、これらの試薬を利用する実験を行います。 <ol><li> （ - 3分〜2）呼吸が停止するまでのイソフルラン〜1ミリリットルで安楽死室で動物を生け贄に捧げます。 </li><li> （ - 10分5）血液が組織からクリアされるまで、心臓内に4℃で灌流液（ 表1）でマウスを灌流するために〜5ミリリットル/分に設定し、蠕動ポンプを使用してください。 </li><li>固定液を灌流液を変更します（ 表1）4℃で首と尾が大幅に補強されるまで（5から10分）。 </li><li>灌流を終了し、動物を刎ねます。慎重に最初の頭蓋骨を周囲の結合組織を除去した後、頭蓋骨の腹面から骨を除去し、20で説明したように、（ 図1a）は、脳を持ち上げて、頭蓋骨から脳を解剖します。 注：小脳の片葉小節のローブと嗅球を周囲の骨の除去に特別な注意を与えます。 <ol><li>優しく脊髄と脊柱の間の微妙な、鋭いハサミの先端を挿入し、21で説明したように、離れて個々の脊柱セグメントを切断することにより脊柱から脊髄を解剖。 </li></ol></li><li>プラスチックマウス脳マトリックス中に配置し、マトリクスの刃で正中線に沿って切断することにより脳を二等分します。 </li><li> 50ミリリットルの遠心分離槽を分離する各半球および脊髄を追加ヒドロゲル溶液（ 表1）~40 mlのエス。この点から前方のすべての後続のステップ中に蛍光団を保護するためにアルミホイルでチューブをカバーしています。 </li><li>穏やかに7日間（〜30 rpm）で振とうしながら4℃でヒドロゲル中にサンプルを含むチューブをインキュベートします。 </li></ol> 2.重合<ol><li>遠心管にサンプルと〜ヒドロゲルの25ミリリットルを維持し、ヒドロゲルの〜25ミリリットルを廃棄します。 </li><li>キャップを外し、デシケータージャーに遠心管を配置し、真空に瓶を接続することにより、試料を脱酸素。溶液およびフォームの泡の出てくるために溶存ガスを可能にするために、少なくとも10分間真空を適用します。静かに気泡を取り除くために振ります。 </li><li> 3分 - 2を超える100％の窒素ガスでデシケータージャー内に真空を交換してください。圧力が（デシケータージャーのトップを開くことができたら）等化したら、すぐに酸素の再導入を防止するためのチューブをキャップ。 </li><li>ポリマー重合が完了するまで、3時間37℃の水浴中で試料と管を配置することによってヒドロゲルをIZE。重合ヒドロゲルは、ゲル状のコンシステンシー（ 図1b）にかかります。注意：上部の未重合ヒドロゲルの少量が許容されます。 </li><li>サンプルから過剰なヒドロゲルを切り取った後、遠心分離管から重合サンプルを除去するために、へらを使用します。 </li><li>無料のワイプリントにサンプルを置き、静かにサンプル上の重合ヒドロゲルの薄層だけを残し、擦れ、その上に組織を圧延することにより、残りのヒドロゲルを削除します。 </li></ol> 3.脂質除去注：脂質クリア組織は壊れやすいので、取り扱いに注意して。チューブを排出する際にサンプルが失われないようにセルストレーナーを使用してください。また、試料は、屈折率整合プロセス中に逆転させることができる膨張が、クリア処理を通じて膨潤します。 注意：SODIUMドデシル硫酸ナトリウム（SDS）およびホウ酸が刺激されています。ドラフト内で、適切な個人用保護具（白衣、手袋、目の保護）とそれらを利用した実験を行います。 <ol><li>きれいな50ミリリットルの遠心分離管に重合したサンプルを移し、緩衝液（ 表1）をクリアする45ミリリットルを追加します。穏やかに振とうしながら45℃（〜30 rpm）でインキュベートします。化学廃棄物への使用クリアリングバッファをオフに注ぐ、新鮮なクリアリングバッファの〜45ミリリットルを添加することにより、最初の7日間毎日バッファを変更します。蛍光団を保護するためにアルミホイルで覆われたサンプルを含む遠心管を保管してください。 </li><li> 3日 - 最初の7日後、すべての2〜40°Cと交換決済バッファーでサンプルをインキュベートします。すべての脂質が可溶化され、組織は透明（ 図1c）に達するまでクリアし続けます。マウス脳半球のための6週間と2 - - 脊髄のための4週間これは4になります。 </li><li>組織がクリアされると、転送新しい50mlの遠心管に試料及び残留SDSを除去するために、24時間にわたって穏やかに振盪（約30 rpm）しながら40℃でPBST（ 表1）で4回洗浄します。 </li></ol> 4.分子染色注意：4 &#39;、6-ジアミジノ-2-フェニル二塩酸塩（DAPI）は、刺激性であり、それを利用した任意の実験はドラフト内で、適切な個人用保護具（白衣、手袋、目の保護）を用いて行われるべきです。 <ol><li>きれいな15mlの遠心チューブにサンプルを移し、1×PBST、pH7.5中に1μg/ mlのDAPIで埋めます。 24時間室温（RT）でインキュベートします。蛍光団を保護するためにアルミホイルで覆われたサンプルを含む遠心管を保管してください。 </li><li>少なくとも6時間/洗浄を室温でPBSTで3回洗浄します。 </li></ol> 5.屈折率整合注意：SH 2,2&#39;-チオジエタノール（TDE）は、刺激性であり、任意の実験は、それを利用しますドラフト内で、適切な個人用保護具（白衣、手袋、目の保護）を行うことがウルド。 <ol><li>きれいな15ミリリットルの遠心分離管にサンプルを移し、1×PBS、pH7.5中30％（v / v）のTDEで満たします。 24時間穏やかに振とうしながら40℃（〜30 rpm）でインキュベートします。 </li><li> 1×PBS、pH7.5中63％（v / v）のTDEとの交流30％TDE。穏やかに24時間振盪しながら40℃でインキュベートします。 注：サンプルは、ほぼ元の大きさに戻って縮小します。 </li><li>平らな、きれいな表面上のサンプルの厚さよりもわずかに大きい均一な直径を持つシリンダーに再利用可能な接着剤の一部をロールバックします。 </li><li> 40ミリメートルのガラスボトムディッシュにオープンエンド円（上部に小さな開口部を持つ〜25ミリメートル、内径）で再利用可能な接着剤シリンダーを置きます。訂正がなされる必要がある場合は、ガラスからシリンダーを取り外し、カミソリの刃で切断しました。 </li><li>によって、再利用可能な接着剤とガラスとの間にシールを作成するために、P1000のピペットチップを使用してシリンダーの外側リムの周りにそれを転がします。 </li><li>場所〜100μlの63％のガラス皿の上にTDEと再利用可能な接着剤の円内のガラス表面を覆うように周りに広がります。 </li><li>へらを使用して、慎重に気泡を形成しないように注意しながら、円の真ん中にサンプルを配置します。 </li><li>サンプル上に直接63％TDEの別の100μLをピペットで、その後すぐに再利用可能な接着剤の上に40ミリメートルの円形のカバーガラスを配置します。 </li><li>カバーガラスが試料と接触したまでインデックス、中央、及び両手の薬指を使って、慎重に円形の周囲を押します。 </li><li>ゆっくりと、表面に支え直立位置にガラスボトムディッシュをもたらすソリューションは、上部に小さな開口部に到達するまで、ピペットを用いて63％のTDEを追加します。無料のソリューションは、ガラス上に滴下しないようにピペットチップをオフに乾燥するためにワイプ糸くずを使用してください。チャンバー内の気泡を回避するために、ピペットのすべての方法を空にすることはありません。 </li><li>に小さな開口部は、円を囲み、閉鎖チャンバーを作成するために、シリコンエラストマーを追加します。それは撮影前に乾燥するのを5分待ってください。 </li></ol> 6.顕微鏡注意：共焦点、単一平面照明と光シート顕微鏡で使用されるレーザーは非常に強力であり、失明を引き起こす可能性があります。レーザーを利用した任意の実験は、広範囲の訓練の後、大きな注意し、適切な眼の保護を行うべきです。 <ol><li>レーザー走査共焦点顕微鏡のステージ上の内部サンプルとイメージング室を配置します。 </li><li>イメージングソフトウェアを開きます（材料の表を参照してください）​​。アルゴン励起レーザーをオンにするには、その後、プログラムのレーザアイコンの上部にある[設定]タブをクリックします。レーザーの電源を入れて、30％にスライドスケールを移動させるために、アルゴンの横にあるボックスをチェックします。選択された位置まで温め、ゆっくりとポインタを守ってください。 <ol><li>上部の取得]タブに戻ります。大きなボックスに「ビーム経路の設定＆＃34; /負荷に行くボックスを設定保存し、画像YFP（527 nm）のに適した波長チャネルを選択するドロップダウンメニューを選択します。 </li></ol></li><li>チャネルの設定]ボックスの下：「現在のオブジェクトの目的」というラベルの付いた赤いハイパーリンクを配置することにより、イメージングのために適切な目標を選択します。タブをクリックすると、すべての利用可能な目標を開き、選択がタレットを自動的に回転します。 <ol><li>使用目的（ 例えば 、10倍）大きい作動距離（組織の厚さよりも大きいが、画像化される、 例えば11 mm）とし、大きな開口数（ 例えば 、0.3）を有する面内画像の解像度を最大にし、最小限にします光学部の厚さ。 </li></ol></li><li> 1,024×1024または目的の横方向の解像度の限界に適した値に、「フォーマット」と呼ばれる、取得行列を設定するには：「解像度XY」ウィンドウのドロップダウンメニューの左の欄には、開きます。 （可能な限り低いズーム倍率を設定し、例えば 、1。7）。 </li><li>同じウィンドウでは、それに応じて個別に標識されたメニューをドロップダウンすることにより、8ラインの平均値と1フレーム平均パラメータを設定します。高い信号対雑音比と迅速な取得のための小さな値の大きい値を使用してください。 注：8ラインの平均値と1フレームの平均は約4時間で、マウスの脳半球の高品質な画像を取得します。 </li><li>ステージコントロールを使用してサンプルを探します。代表的なフィールドが表示されたら、ゲインとオフセットを設定します。 フルオロフォア、ゲインとオフセットは、組織の種類に基づいて、実質的に変化し、および/または画像化される解剖学的特徴：注意してください。 YFPの場合、オフセット用のゲインおよび-1.0から-1.5まで、760から780に値を設定します。 </li><li> 「タイルスキャン」を選択し、関心領域の境界を選択します。 zスタックの設定上限値と下限値を取得します。目的の光学部の解像限界に基づいて、光学部の厚さを設定します。 注：目的の光学部の解像限界は、主に、その開口数によって定義されます。ほとんどの顕微鏡制御ソフトウェアが自動的に使用されている目的のために適切な値を計算します。約11ミクロンとなる0.3の開口数の10倍の対物レンズのため。 </li><li>取得を開始します。 注：これは、複数の時間がかかることがあります。 </li><li>関心の特定の領域から高倍率の対物レンズを使用して、より高い解像度の画像を収集します。 </li></ol>

<ol>
	<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+tomography:+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits.">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li><li>Kleinfeld, D., 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=Large-scale+automated+histology+in+the+pursuit+of+connectomes.">Large-scale automated histology in the pursuit of connectomes.</a> J Neurosci. 31, 16125-16138 (2011).</li><li>MacKenzie-Graham, A., 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+multimodal,+multidimensional+atlas+of+the+C57BL/6J+mouse+brain.">A multimodal, multidimensional atlas of the C57BL/6J mouse brain.</a> J Anat. 204, 93-102 (2004).</li><li>Denk, W., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upmanship:+why+multiphoton+imaging+is+more+than+a+gimmick.">Photon upmanship: why multiphoton imaging is more than a gimmick.</a> Neuron. 18, 351-357 (1997).</li><li>Denk, W., 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=Anatomical+and+functional+imaging+of+neurons+using+2-photon+laser+scanning+microscopy.">Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.</a> J Neurosci Methods. 54, 151-162 (1994).</li><li>Zipfel, W. R., Williams, R. M., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+magic:+multiphoton+microscopy+in+the+biosciences.">Nonlinear magic: multiphoton microscopy in the biosciences.</a> Nat Biotechnol. 21, 1369-1377 (2003).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2, 932-940 (2005).</li><li>Theer, P., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+fundamental+imaging-depth+limit+in+two-photon+microscopy.">On the fundamental imaging-depth limit in two-photon microscopy.</a> J Opt Soc Am A Opt Image Sci. Vis. 23, 3139-3149 (2006).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Erturk, A., 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Hama, H., 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Susaki, E. A., 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Spence, R. D., 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=Bringing+CLARITY+to+gray+matter+atrophy.">Bringing CLARITY to gray matter atrophy.</a> Neuroimage. 101, 625-632 (2014).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9, 1682-1697 (2014).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Costantini, I., 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+versatile+clearing+agent+for+multi-modal+brain+imaging.">A versatile clearing agent for multi-modal brain imaging.</a> Sci Rep. 5, 9808 (2015).</li><li>Zheng, H., Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+CLARITY+for+visualizing+immunofluorescence+labeling+in+the+developing+rat+brain.">Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain.</a> Brain Struct Funct. , (2015).</li><li>Parra, S. G., 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=Multiphoton+microscopy+of+cleared+mouse+brain+expressing+YFP.">Multiphoton microscopy of cleared mouse brain expressing YFP.</a> J Vis Exp. (67), e3848 (2012).</li><li>Weinger, J. G., 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=Two-photon+imaging+of+cellular+dynamics+in+the+mouse+spinal+cord.">Two-photon imaging of cellular dynamics in the mouse spinal cord.</a> J Viz Exp. (96), e52580 (2015).</li><li>Hama, H., 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=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nat Neurosci. 18, 1518-1529 (2015).</li><li>Treweek, J. B., 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=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nat Protoc. 10, 1860-1896 (2015).</li></ol>

The authors have nothing to disclose.

ハイドロゲル包埋組織（パッシブCLARITY）の受動クリアは組織の大部分をクリアするための簡単​​で安価な方法です。このアプローチは、専用の設備を必要とせず、容易に温度制御シェーカー中で行うことができます。数週間のスパンで、このような脳全体または脊髄などであっても大規模な、非常に有髄組織は、顕微鏡検査のために透明性と適切ななります。このレポートは、CNS組織の清...

The ability to image complete neuroanatomical structures is immensely valuable for understanding the brain in health and disease. Traditionally, 3D imaging has required tissue sectioning to provide the axial resolution and to visualize deep anatomical structures. This approach can produce high-resolution data sets, but requires sophisticated image reconstruction techniques and is very labor intensive. As a result, it has been limited to the imaging of small volumes of tissue1-3. Optical sectioning, on the other hand, is well suited for the creation of high-resolution 3D images of fluorescently labeled tissues. Since optical sectioning is inherently three dimensional, it does not require extensive computation to produce a 3D image volume. However, light scattering and tissue opacity limit the depth at which tissues can be optically sectioned. The depth of imaging is limited to about 150 &#181;m in laser scanning confocal microscopy and to less than 800 &#181;m using two-photon excitation microscopy4-8.
In order to overcome these limitations, several optical clearing techniques have been recently developed and then further refined to permit the deep microscopic imaging of intact tissues. The use of benzyl alcohol and benzyl benzoate (BABB) to render fixed tissues transparent was among one of the earliest approaches9. However, this approach was limited by the quenching of fluorescence in the samples and incomplete clearing of highly myelinated structures10. Refinements of this technique, such as 3D Imaging of Solvent Cleared Organs (3DISCO), have led to very rapid and complete tissue clearing, but still suffer from rapid loss of fluorescent signals, especially yellow fluorescent protein (YFP)10. Water-based clearing solutions, such as Sca/e11 and SeeDB12, preserve fluorescent signals, but do not completely clear highly myelinated tissues. Clear, Unobstructed Brain Imaging Cocktails and Computational analysis (CUBIC) is a promising new optical clearing technology that appears to overcome the limitations of previously developed water-based clearing solutions13. In contrast with other optical clearing techniques, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY)14 embeds the brain in a porous matrix that provides structural integrity to proteins, nucleic acids and small molecules, while leaving lipids unbound. The lipids are then removed electrophoretically, culminating in an optically cleared brain that can be easily visualized and equally easily probed using commonly available techniques.
Electrophoretic tissue clearing (ETC) is used to remove lipids from hydrogel embedded tissues in CLARITY, however, ETC can be difficult to implement consistently and tissues subject to electrophoresis can exhibit tissue distortion, browning, loss of fluorescence and antibody reactivity. Passive lipid clearing avoids these limitations, but requires increased clearing times15-17. Passive clearing also permits the clearing of large numbers of samples in parallel, since it is not limited by the number of ETC apparatuses available. Decreasing the concentration of paraformaldehyde and excluding bis-acrylamide from the hydrogel have led to great increases in the speed of clearing at the expense of greater tissue expansion16. Less expensive alternatives to the refractive index matching solution used in the original CLARITY technique have been developed17-19. 2,2'-thiodiethanol (TDE) is an inexpensive and rapid brain clearing agent that reverses some of the tissue expansion that occurs during lipid removal18. This report incorporates passive clearing of hydrogel embedded tissue and the use of TDE as a refractive index matching solution to the original CLARITY technique to produce a highly reproducible and inexpensive protocol for the optical clearing of the mouse central nervous system.

<table><tbody><tr><td>10x phosphate buffered saline (PBS)</td><td>Fisher</td><td>BP399-1</td><td>buffers</td></tr><tr><td>32% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15714-5</td><td>perfusion and hydrogel</td></tr><tr><td>2,2&#039;-thiodiethanol (TDE)</td><td>Sigma-Aldrich</td><td>166782-500G</td><td>refractive index matching solution</td></tr><tr><td>40% acrylamide</td><td>Bio-Rad</td><td>161-0140</td><td>hydrogel</td></tr><tr><td>2% bis-acrylamide</td><td>Bio-Rad</td><td>161-0142</td><td>hydrogel</td></tr><tr><td>VA-044 initiator</td><td>Wako Pure Chemical Industries, Ltd.</td><td>VA044</td><td>hydrogel</td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B7901</td><td>clearing buffer</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166-5</td><td>clearing buffer</td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>55255-1</td><td>buffers</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>52002-100G</td><td>preservative</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500G</td><td>buffers</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393-50KU</td><td>perfusion</td></tr><tr><td>Sodium nitrate</td><td>Fisher</td><td>BP360-500</td><td>perfusion</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>nuclear stain</td></tr><tr><td>99.9% isoflurane</td><td>Phoenix</td><td>57319-559-06</td><td>anesthetic</td></tr><tr><td>Hydrocholoric acid</td><td>Fisher</td><td>A1445-500</td><td>buffers</td></tr><tr><td>Glass bottom dish</td><td>Willco</td><td>HBSB-5040</td><td>Willco dishes</td></tr><tr><td>Reusable adhesive</td><td>Bostick</td><td>371351</td><td>Blu-Tack</td></tr><tr><td>Silicon elastomer</td><td>World Precision Instruments</td><td>KWIK-CAST</td><td>Kwik-Cast</td></tr><tr><td>Lint-free wipe</td><td>Kimberly-Clark</td><td>34120</td><td>KimWipe</td></tr><tr><td>Mouse brain matrix</td><td>Roboz</td><td>SA-2175</td><td>sectioning tissue</td></tr><tr><td>Matrix blades</td><td>Roboz</td><td>RS-9887</td><td>sectioning tissue</td></tr><tr><td>Peristaltic pump</td><td>Cole-Parmer</td><td>77122-24</td><td>pefusion</td></tr><tr><td>Laser scanning confocal microscope</td><td>Leica</td><td>SP5</td><td>microscopy</td></tr><tr><td>Imaging software</td><td>Leica</td><td>LAS AF</td><td>microscopy</td></tr></tbody></table>

optical clearing of the mouse central nervous system using passive clarity

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

脳の構造組織の可視化形態との接続が健康と病気で脳機能にどのような影響を与えるかを理解するために重要です。光学決済技術は、私たちが凝集的形態との接続性を研究することができ、無傷の組織における3次元の画像細胞集団にことを可能にします。 細胞の亜集団のための特異的プロモーターによって駆動される蛍?...

Watch this Scientific Journal Video about Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY at JoVE.com

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

パッシブCLARITYを使用したマウス中枢神経系の光学クリア

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

optical-clearing-mouse-central-nervous-system-using-passive

Research

JoVE Journal

Neuroscience

13.4K ビュー.  University of California, Los Angeles. Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

ビデオ: Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

YFPを発現クリアマウス脳の多光子顕微鏡

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

神経科学

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

ビジュアル欠陥を特徴づけるために慢性高眼圧症のレーザー誘起マウスモデル

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

医学

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

共焦点光シート顕微鏡全体マウスの脳のミクロンスケールの分解能光トモ

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

CLARITY / CUBIC技術を用いたマウスの脊髄にセロトニン繊維のイメージング

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

麻痺、視神経脊髄炎自己抗原アクアポリン 4 の T 細胞特定のマウスの視覚系障害の誘導

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

免疫・感染学

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

新規パッシブ全体の中枢神経系組織における光透過性の急速な生産のための方法をクリア

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

CLARITY組織クリアリング法による全マウントレティナの免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

CLARITY組織クリアリングによるインタクトニューロンデンドライトのイメージングと定量化

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

メゾスコピックから顕微鏡スケールまでのニューロンイメージングのための組織クリアリング法

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

光干渉断層撮影法の網膜症モデルマウスへの応用

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...