The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

倫理の声明：承認されたプロトコルとメモリアルスローンケタリングがんセンター（MSK）施設内動物管理使用委員会（IACUC）の倫理ガイドラインの下に従って行った説明in vivo動物実験のすべて。 噴霧装置の1建設<ol><li> 2本のネジ（×25 mm 2と4）を使用して、パートB（12.7 X 10.7 X 1.8センチメートル3）の中央に直立木質部A（12.5 X 2.5×1.8センチメートル3）を取り付けます。 1本のネジを使用して、パートA（12.5 X 2.5×1.8 cm 3）での中央に木部C（11×2.5×1.8 cm 3で）を取り付け、その結果、C部（1.8×11×2.5cm 2の3）はまだ移動することができます。 図1を参照してください。 </li><li>二つのループ、どちらかに1を形成するために介して2つの穴のプラスチック3オンスミニ噴霧器（D）のスプレーボトルトリガの下部先端のドリルスルーとステンレス棒をプッシュ（1/16 &quot;鋼10センチ）（E）トリガーの側面。 </l私&gt; <li>抜けケーブルタイを防止するために、ダクトテープ（F）でスプレーボトルの底部を包みます。 2プラスチック製のケーブルタイを使用して、木質部C（11のx 2.5×1.8センチメートル3）（28センチメートル）（G）へのスプレーボトルを取り付けます。 </li><li> 011サーボモータ（I）を遮断し、サーボ制御（H）のケーブルが緩んでいないでそれを再接続します。そして、ダクトテープを使用して、木質部A（12.5 X 2.5×1.8センチメートル3）の上部にサーボモータを接続します。 </li><li>ペーパークリップ（K）を使用して、サーボモータレバーに鉛筆（J）を取り付けます。しっかりとプラスチックカバーされツイスト線（L）を使用して、スチールロッドループに鉛筆の最も外側の部分を接続し、ダクトテープと鉛筆の端を固定します。 </li><li>サーボモータ制御装置の磁気ケーブルコネクタ（M）を遮断し、スピーカーケーブル（N）に再接続します。そして、木質部B（12.7 X 10.7 1.8×cm 3）とするサーボモータ制御装置をテープ。半分に磁気コネクタ付きW1ケーブルをカットし、銅sの緩い最後に1部を添付ピーカーケーブル（1メートル）。 （磁気）12トグルスイッチとp1の電源が利用できW1ケーブルと9Vバッテリーに接続します。 </li></ol>法の2.感度決意<ol><li> 1.5 mLのマイクロ遠心チューブでは、260μgの（347ナノモル）、52μgの（69ナノモル）、26μgの（34ナノモル）の量で逆浸透水（100μL）の[3のRu（bpyで）]のCl 2の溶液を調製、5μgの（6.9ナノモル）、3μgの（3.5ナノモル）、520 ngの（694ピコモル）、260 ngの（347ピコモル）、52 ngの（69ピコモル）、26 ngの（34ピコモル）、5 ngの（6.9ピコモル）、および3 ngの（3.5ピコモル）。 </li><li>各100μLをミックス[のRu（bpyで）3]顕微鏡スライド上の硝酸セリウムアンモニウムの水溶液100μL（（NH 4）2 Ceの（NO 3）6）の水（25 mM）のでのCl 2溶液。 </li><li> イメージングソフトウェアを初期化することにより、生物発光リーダーに買収を設定します。 <ol><li>ユーザプロファイルにサインインし、アックイを探しますジションコントロールパネル。 「初期化」をクリックすると、機器の準備ができるまで待ちます。 </li><li>チェックされ、「画像表示モード」を探し、「発光」と「写真」がチェックされていることを確認し、その「蛍光」。 </li><li> 20秒に「発光」の設定「露出時間」に変更します。 「発光」のための残りの設定を設定し、次のように：「ビニング」：中。 「F /ストップ」：1。そして、「発光フィルタ」：オープン。 注：露光時間は、提示さ設定と異なる場合は、使用機器および実験設定に応じて適応させることが必要になる場合があります。 </li><li> 「露出時間」：次の設定を使用」、写真」のためのオート。 「ビニング」：ミディアム。および「F /ストップ」：8.「フィールドビューの「ドロップダウンメニュー用のtarget.Lookを画像化するに従って、「件名の高さ」を調整してください。初期設定は「C」でありますCとの間の「B」に変更します（14センチメートル距離アメーラと試料ステージ）。 </li></ol></li><li> 酸化剤から保護するために撮影室の床に黒い画用紙のシートに顕微鏡スライドを配置することによって、噴霧器を設定します。 [のRu（bpyで）3]の100μLdroplet（NH 4）の水溶液100μLでのCl 2 -溶液を混ぜる2のCe（NO 3）6.緑色のボックスの十字線を観察します。 <ol><li>関心領域は、試料ステージ上に表示される緑色のライトボックスの十字線の中央にあるように、黒画用紙に被写体を配置します。木製のサポートから、プラスチック製のスプレーボトルを取り外して噴霧器を準備します。そのプラスチック製のリザーバーに、木製のサポートにそれを再接続します（水/エタノール中3 1）のトリエチルアミンの溶液を埋めます。 </li><li>生物発光リーダーの内部噴霧器を配置し、電源コードがネブライザーコードから切り離されていることを確認します。その電源スイッチを確認してください上で、トグルスイッチがオフになっている、と赤色のLEDが点灯します。スプレーノズルヘッドによる被写体に向けてカメラからの眺め閉塞を最小限に抑えながら、噴霧流は、被写体上の関心領域に向かって指摘されるように噴霧器を置きます。 </li><li>スプレーからそれらを保護するために、任意の潜在的なホットスポット（顕微鏡スライドまたは注射部位に例えば、白マーク）の上に画用紙の小さな黒い駒を置きます。場所が撮像対象、噴霧器、または磁気ドアラッチと干渉しないように、撮像チャンバ内部ネブライザ遠隔コードの少なくとも40センチ、。イメージングシステムのドアを閉じます。 注：十字線がで設定」ビューのフィールド」に基づいてサイズを変更します」リビングイメージ; &quot;これは「B」に設定されていることを確認してください。 </li></ol></li><li>撮像シーケンスを開始することにより画像を取得します。で、「取得」をクリックして「取得コントロールパネル。」第1の撮像sequ上レンスは、必要に応じて自動保存を有効にする（推奨）とデータフォルダを選択します。シーケンスの最後まで「編集イメージングラベル」ダイアログを無視します。 注：制御ソフトウェアは、機器のアクションは、ステップバイステップでリアルタイムに表示されます。測定を調製し、正しい位置に試料ステージを移動した後、それはカメラのシャッターを開き、測定時間をカウントします。シャッター開口部は、機械によって生成され、クリック音によって聞くことができます。 </li><li>化学発光を生成するために、トグルスイッチを3回切り替えることにより：シャッターが開くと、トリエチルアミンの溶液の3バースト（水/エタノールで3、スプレーバーストあたり0.24±0.04 mLの1）をスプレー。 注：試料ステージは、測定中に移動します。これを可能にするために、機器の内部に十分な（最小40センチメートル）ケーブルを残します。解決策は、上昇管によって熱望することができ、噴霧器によって噴霧されることと配管内に気泡がないことを確認します。いくつかの予備バッテリー含みを持っています必要な場合には準備ができてネブライザーのための。 </li></ol>全身静脈内注射後のin vivoイメージング3 <ol><li> 1.5 mlのマイクロ遠心管中で、[Ruの（BPY）3] Cl 2をナノモル8〜33を含有するリン酸緩衝生理食塩水（PBS）溶液100μLを調製します。同時に、（NH 4）の水溶液2のCe（NO 3）水に6（25 mm）を準備します。 </li><li>静脈内に健康なマウスの尾静脈に[のRu（bpyで）3] Cl 2を100μLを注入した（n = 5）。 </li><li> CO 2窒息を介してマウスに注射後10分を安楽死させます。 <ol><li>心臓や肺を露出するようにU字状に肋骨弓を削除した後、胴体からYカットで皮膚を取り除きます。右心房にコンセントを切断し、左心室18に24ゲージの針を介してPBSの20mLのを注入することにより、マウスを灌流。慎重に腹を切って皮膚や腎臓および肝臓を露出させます。目に見えるカットを作成するために臓器を通って長手方向にカットします。 </li></ol></li><li> 以下の変更を行い、ステップ2.3から2.6に記載されているように買収を設定します。 <ol><li>十分ネブライザプラスチックリザーバを洗浄した後、代わりにトリエチルアミン水（NH 4）2の溶液のCe（NO 3）6（25ミリモル）でそれを埋めます。 注：徹底的に（NH 4）2 Ceの（NO 3）6は、いくつかの使用後にスプレーノズルを破壊する可能性を結晶化するので、使用するたびに、ネブライザーノズルを洗浄することが重要です。 </li></ol></li><li> 動物全体または器官のサンプルは、イメージングのために使用します。 <ol><li>腹部全体イメージングのために、カメラ、機器の背面を指して頭に直面して開いた腹部とマウスの死体を置きます。緑色のライトボックスの十字線で画像化される臓器（ 例えば、肝臓や腎臓）を中央に配置します。 </li><li> Fまたは臓器イメージングおよび定量個々の、イメージング装置からマウスを削除し、それを突き止めます。すでにオープンした体腔から出発し、内部器官（ 例えば、腎臓、肝臓、肺、筋肉、脾臓、脳、および心臓）を切り出します。筋肉組織を切除するために後肢の皮膚を介してカットします。慎重に脳を切除するメスで頭蓋骨を開きます。 <ol><li>関心のある器官は、肝臓、腎臓、脾臓の場合は、縦方向に半分にすべての器官をカットし、黒画用紙のシャーレまたは片に各臓器を置きます。 </li></ol></li><li>単一臓器のための化学発光トレーサーの相対発光を確立するためのステップ2.3から2.6に記載されている手順に従ってください。 </li></ol></li></ol>リンパ節のin vivoイメージング4. <ol><li> [のRu（bpyで）3] Cl 2を80ナノモルを含むPBS溶液10μLを準備します。水溶液（NH 4）2 Ceと準備（NO 3）6ワシントン州同時にター（25 mM）の。 </li><li>健康なマウスの後肢に皮下溶液10μLを注入した（n = 5）。陰性対照として、純粋なPBSの10μLと反対側の足を注入。 15分注射後、CO 2窒息を介してマウスを生け贄に捧げます。膝窩リンパ節までのリンパ運河を露出するために、両方の後ろ足の皮膚を取り除きます。 </li><li>ステップ3.4で説明したように買収を設定します。 </li><li>定量化のために、両方の後肢からの膝窩リンパ節を除去半分にそれらを切断し、ペトリ皿上に酸化剤とそれらをスプレー、前述したように（ステップ3.5.3）。 </li></ol>

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Vol 13. Scandium, Yttrium, Lanthanum, and Lanthanide Nitrates. , (1983).</li><li>Reiner, T., Jantke, D., Marziale, A. N., Raba, A., Eppinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-Conjugated+Affinity+Labels:+A+New+Concept+to+Create+Enantioselective+Artificial+Metalloenzymes.">Metal-Conjugated Affinity Labels: A New Concept to Create Enantioselective Artificial Metalloenzymes.</a> ChemistryOpen. 2, 50-54 (2013).</li><li>Zanarini, 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=Synthesis+and+Electrochemiluminescence+of+a+Ru(bpy)3-Labeled+Coupling+Adduct+Produced+on+a+Self-Assembled+Monolayer.">Synthesis and Electrochemiluminescence of a Ru(bpy)3-Labeled Coupling Adduct Produced on a Self-Assembled Monolayer.</a> J. Phys. Chem. C. 112 (8), 2949-2957 (2008).</li><li>Liu, R., Lv, Y., Hou, X., Yang, L., Mester, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Quantitation+Using+Ru-NHS+Ester+Tagging+and+Isotope+Dilution+High-Pressure+Liquid+Chromatography-Inductively+Coupled+Plasma+Mass+Spectrometry+Determination.">Protein Quantitation Using Ru-NHS Ester Tagging and Isotope Dilution High-Pressure Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry Determination.</a> Anal. Chem. 84 (6), 2769-2775 (2012).</li><li>Jantke, 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=Synthetic+strategies+for+efficient+conjugation+of+organometallic+complexes+with+pendant+protein+reactive+markers.">Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers.</a> J. Organomet. Chem. 744, 82-91 (2013).</li><li>Aoki, Y., 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=An+experimental+xenograft+mouse+model+of+diffuse+pontine+glioma+designed+for+therapeutic+testing.">An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing.</a> J Neurooncol. 108 (1), 29-35 (2012).</li><li>Forster, R. J., Bertoncello, P., Keyes, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenerated+Chemiluminescence.">Electrogenerated Chemiluminescence.</a> Annual Rev. Anal. Chem. 2, 359-385 (2009).</li><li>Connell, T. U., James, J. L., White, A. R., Donnelly, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Labelling+with+Versatile+Phosphorescent+Metal+Complexes+for+Live+Cell+Luminescence+Imaging.">Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.</a> Chem. Eur. J. 21 (40), 14146-14155 (2015).</li><li>Zhou, X., 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=Synthesis,+labeling+and+bioanalytical+applications+of+a+tris(2,2&#8242;-bipyridyl)ruthenium(II)-based+electrochemiluminescence+probe.">Synthesis, labeling and bioanalytical applications of a tris(2,2&#8242;-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.</a> Nat. Protoc. 9 (5), 1146-1159 (2014).</li><li>B&#339;uf, 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=Encapsulated+Ruthenium(II)+Complexes+in+Biocompatible+Poly(d,l-lactide-co-glycolide)+Nanoparticles+for+Application+in+Photodynamic+Therapy.">Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.</a> ChemPlusChem. 79 (1), 171-180 (2014).</li><li>Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+its+applications+in+surgery.">Nanotechnology and its applications in surgery.</a> Brit. J. Surgery. 97 (4), 463-465 (2010).</li><li>Barry, N. P. E., Sadler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+for+Metals+in+Medicine:+How+Nanotechnology+May+Help+To+Shape+the+Future.">Challenges for Metals in Medicine: How Nanotechnology May Help To Shape the Future.</a> ACS Nano. 7, 5654-5659 (2013).</li><li>Hasan, K., Bansal, A. K., Samuel, I. D. W., Rold&#225;n-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Emission+of+Cationic+Iridium+(III)+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand.">Tuning the Emission of Cationic Iridium (III) Complexes Towards the Red Through Methoxy Substitution of the Cyclometalating Ligand.</a> Nat.Sci. Rep. 5, 1-15 (2015).</li><li>Truong, J., 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=Chemiluminescence+detection+with+water-soluble+iridium(III)+complexes+containing+a+sulfonate-functionalised+ancillary+ligand.">Chemiluminescence detection with water-soluble iridium(III) complexes containing a sulfonate-functionalised ancillary ligand.</a> Analyst. 139 (22), 6028-6035 (2014).</li></ol>

The authors have nothing to disclose.

ここでは、光学的、化学発光レポーターによって作成された光子の放出を介して組織の輪郭を描くことのできる技術を提示しています。他のとは対照的に、多くのこの化学発光レポーター系は、非放射性であり、非常に高感度レベルでの検出を容易に画像化プローブを用い、技術4、5、6、7、8?...

ここ数十年では、イメージング技術は、医師が病気を診断し、監視する方法に革命をもたらしてきました。これらのイメージング技術は、しかしながら、例えば、陽電子放射断層撮影（PET）、単一光子放射型コンピュータ断層撮影法（SPECT）、コンピュータ断層撮影（CT）、磁気共鳴イメージング（MRI）などの全身画像化システムに大きく制限されています。特に注意は、がんが注目されている、技術イメージングブレークスルーを大幅にこの病気を診断し、治療される方法を改善しています。手術室：これらの進歩にもかかわらず、これらのイメージング技術がちょうど収まりきらない一つの場所があります。全身イメージング技術は、手術計画に役立つことができますが、それらは一般的に、腫瘍組織の全てが除去された又は残留腫瘍組織を切除縁1に隠されたままかどうかを医師がリアルタイムで決定するのに役立つのに十分に高い空間分解能を欠いています。ノー浸潤がしていることを確認し腫瘍マージンが最も重要な外科的な目標の一つであり、外科医は、厳格かつ慎重な組織切除の間にタイトロープを歩く必要があります残されています。あまりに多くが除去されると、患者に対する望ましくない副作用が悪化します。少なすぎるが削除された場合、再発率は2、3に増加しています。したがって、正確な腫瘍のマージンを描くために重要であり、我々は、化学発光術中イメージングは​​、そうでなければ、確立された技術で検出されない状態になる可能性があり、悪性組織を可視化するために外科医を支援することにより、腫瘍のマージンの識別の精度を向上させるために助けることができると信じています。 現在術中イメージングシステムとしてのそれらの可能な有用性について研究されている多くのイメージング技術があります。これらは、β-およびγ線放出プローブ4、光学蛍光5、ラマン分光法6を含みます</sup&gt; 7、及びチェレンコフ発光8、9。しかし、今日まで、これらのいずれも、標準的な臨床ツールとして確立されていません。光学蛍光イメージングは​​、これまで、これらの技術の中で最も有望であることが判明している、したがって、最も探索されます。既に多くの用途のための貴重なツールであることが示されているが、それには限界がないわけではありません。確かに、その主な欠点は、本質的に自家蛍光生体組織によって生成されたバックグラウンド蛍光です。このバックグラウンド自己蛍光シグナルは、蛍光シグナルの生成に必要な外部光源により、フルオロフォアに加えて、周囲の組織の励起との積です。実用的な観点から、この自己蛍光は、潜在的に手術室では、この技術の有用性を制限することができる低信号対雑音比をもたらすことができます。 校長蛍光画像上の化学発光画像化の利点は、励起光が必要でないことです。その結果、バックグラウンド自己蛍光はありません。化学発光イメージングでは、励起エネルギーは、代わりに、化学的に生成されます。このプロセスには意図されないバックグラウンド信号を生成しないので、より高い信号対雑音比をもたらすことができます。これは、最終的に切除縁のより精密かつ正確に検出される可能性があります。やや驚くべきことに、術中イメージング技術として、このアプローチの有用性は、10未踏のままです。実際、この手法に最も近い例は、マウス11、12、13におけるミエロペルオキシダーゼによるルミノールの酸化です。化学発光生物医学イメージングは​​、したがって、次のような利点を提供できる研究のかなり未踏の領域です：ハイとローバックグラウンド信号が得られる（1）最小限の自家蛍光gher信号対雑音比。 （2）可視光から近赤外までの化学発光放出の調整可能な波長を、 （3）リンカ技術とすでに存在している標的とする生体分子と結合し、官能化化学発光錯体を、対象となる分子イメージングプローブ14の全体のライブラリへのアクセスを提供します。 この証明の原理研究は、ルテニウムベースの造影剤を使用して、生物医学的環境において化学発光画像化の潜在的な有用性を示しています。この化合物の化学発光特性は、よく1960年代半ば15にさかのぼる調査で、研究されています。化学的活性化の際に、薬剤は、医療用イメージングのために適している約600 nmの16の光を生成します。活性化エネルギーは、励起状態の水17 -follで650ナノ秒の寿命を有するに至る酸化還元反応によって提供されますこの励起状態の緩和時に光子の生成によって負っ。特別に設計されたリモートネブライザーの使用を介して、我々は、化合物の両方のエキソビボおよびインビボで検出することができました。最初の実験の結果は、この技術のさらなる調査を示唆し、非常に有望です。

<table><tbody><tr><td>Wood part A (12.5 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>&amp;nbsp;Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part B (12.7 &amp;times; 10.7 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part C (11 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Screws (4 &amp;times; 25 mm)</td><td>Screwfix</td><td>79939</td><td></td></tr><tr><td>Harmon Face Values 3 oz mini sprayer</td><td>Bed, Bath and Beyond</td><td></td><td></td></tr><tr><td>stainless steel rod (10 cm of 1/16&amp;rdquo; steel)</td><td>Metals Depot Int. Inc.</td><td>2192</td><td></td></tr><tr><td>Pencil Classic HB</td><td>Papermate</td><td>58592</td><td></td></tr><tr><td>Paper clip</td><td>Office Depot</td><td>221720</td><td></td></tr><tr><td>speaker cable</td><td>RCA Inc.</td><td>AH1650SN</td><td></td></tr><tr><td>Energizer 9V alkaline battery</td><td>Energizer Holdings Inc.</td><td>EN22</td><td></td></tr><tr><td>Hitech HS-82MG Micro Servo Motor, 3.4 kg/cm output torque @ 6V</td><td>Hitech RCD USA Inc.</td><td>32082S</td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalog Number</td><td>Comments</td></tr><tr><td>28 cm plastic cable ties</td><td>General Electric Inc.</td><td>50725</td><td></td></tr><tr><td>Duct tape</td><td>3M Inc.</td><td>3939</td><td></td></tr><tr><td>littleBits w1 wire</td><td>littleBits Inc.</td><td>w1 wire</td><td></td></tr><tr><td>littleBits p1 power</td><td>littleBits Inc.</td><td>p1 power</td><td></td></tr><tr><td>littleBits i2 toggle switch</td><td>littleBits Inc.</td><td>i2 toggle switch</td><td></td></tr><tr><td>littleBits 011 servo</td><td>littleBits Inc.</td><td>011 servo</td><td></td></tr><tr><td>20 cm plastic covered wire twist ties</td><td>Four Star Plastics</td><td>71TIE8000</td><td></td></tr><tr><td>Tris(2,2&amp;prime;-bipyridyl)dichlororuthenium(II) hexahydrate</td><td>Sigma-Aldrich Inc.</td><td>224758</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Ammonium cerium(IV) nitrate</td><td>Sigma-Aldrich Inc.</td><td>22249</td><td></td></tr><tr><td>Isofluorane</td><td>Baxter Healthcare</td><td>1001936060</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>PBS1</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich Inc.</td><td>T0886</td><td></td></tr><tr><td>Water</td><td></td><td></td><td>Water was purified using a Milipore Mili-Q (R &amp;ge; 18 M&amp;Omega;)</td></tr><tr><td>Female nude (outbred) mice</td><td>Jackson Laboratories</td><td>1929</td><td>age 5 - 6 weeks</td></tr><tr><td>Strain C57BL/6J&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>NU/J male mice at&amp;nbsp;</td><td>Jackson Laboratories</td><td>2019</td><td>age 6 &amp;ndash; 8 weeks</td></tr><tr><td>IVIS 200 bioluminescence reader</td><td>Caliper Live Science</td><td></td><td></td></tr><tr><td>Live Image 4.2 software</td><td>Perkin-Elmer</td><td>128165</td><td></td></tr><tr><td>Microscope slides</td><td>ThermoScientific</td><td>4951PLUS4</td><td></td></tr></tbody></table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

プロトコル項1に記載のネブライザーシステムは、低コストで容易に入手可能な材料から構成することができます。リモートトリガ生物発光リーダー（ 図1）の内部に還元/酸化剤の噴霧用のインセットであることが意図されます。私たちのデザインは、レンズ〜14センチの距離での生物発光リーダー内の噴霧器の安全な操作を可能にします。レンズのない?...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

生物学的設定における生成と観測化学発光のための新しい技術

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

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8.4K ビュー.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

ビデオ: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Synthesis of Luminol

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. The difference in energy between reactants and products is known as the enthalpy of the reaction. This is found by calculating the difference in energies between the products and the reactants, or &#916;H. If &#916;H is positive, the reaction is endothermic; if it is negative, the reaction is exothermic. While chemical reactions are traditionally thought of as a conversion of reactants into products, many reactions take place in multiple steps, forming intermediates. In chemiluminescent reactions, these intermediates transition from a high energy state to the ground state, releasing a photon of light that may be visible as the reaction progresses.
<h4>Atomic Energy Levels</h4>
Niels Bohr, a Danish physicist, proposed the theory that an electron orbiting the nucleus of the atom can occupy only certain orbits or energy levels. An atom with all of its electrons in the lowest possible energy level is said to be at its ground state. When an electron occupies an energy level higher than the ground state, the atom is in its excited state.
A key assumption in Bohr&#8217;s theory is that an electron remains in the ground state until it is disturbed. Thus, an electron is only elevated to an excited state by absorbing energy. When the electron relaxes back down to the ground state, it releases that energy. Often, this is in the form of a photon of light, whose wavelength is directly related to the energy difference between the excited and ground states.
When an electron is excited to a higher energy level by absorbing light of a certain energy or wavelength, the phenomenon is called fluorescence. This effect can be seen if you expose a laundered white T-shirt to a black light or UV-light. The characteristic glow is caused by the fluorescence of the detergent and bleaching compounds in the shirt, which make it look white under regular light.
<h4>Chemiluminescence</h4>
In a chemiluminescent reaction, an electron is excited by absorbing heat that is released during a reaction. Then, light is released when the electron relaxes back down to the ground state. The major difference between chemiluminescence and fluorescence is that the energy that excites the electrons in chemiluminescence comes directly from the reaction.
One practical application of chemiluminescence is in commercial glow sticks. A glow stick contains two separate solutions: one containing peroxide and the other containing diphenyl oxalate and a color dye. When the two solutions are mixed, which occurs upon activating the glow stick, the resulting reaction between the peroxide and diphenyl oxalate produces energy that excites the dye to a higher energy state. As the reactants are exhausted, the dye returns to the ground state and releases light. This is what gives glow sticks their characteristic color.
<h4>Luminol</h4>
Luminol is a chemical that exhibits chemiluminescent properties and is utilized in a wide range of applications, most notably in forensics. Luminol (C8H7N3O2) emits blue when it is mixed with an oxidizing agent. In the case of forensics, luminol reacts with the iron in hemoglobin, enabling forensic scientists to identify very small traces of blood.
Luminol is synthesized by the dehydration reaction of 3-nitrophthalic acid with hydrazine. The reaction is heated to remove water, and triethylene glycol is added to further increase the temperature. The nitro group of the 3-nitrophthalhydrazide is then reduced using sodium dithionite to form an amino group at high pH. In basic conditions, the 3-nitrophthalhydrazide is soluble. The addition of glacial acetic acid precipitates the luminol.
With potassium hydroxide, luminol forms a dianion. The hydroxide anions deprotonate the two hydrogens that are attached to the nitrogens in the luminol. Oxygen gas oxidizes luminol to its excited state. As it relaxes back to the ground state, it releases a blue-white light.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2015). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Concept

ルミノールの合成

Chemical reactions can be classified as endothermic or exothermic depending on whether the reactions absorb or release heat when they change to products. ...

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化学

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫・感染学

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

医学

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

その場で測定と細胞密度の相関、発光細菌の発光

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生化学

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 ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

BRET歩：バインドされていない励起による蛍光発光から（燃料）

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

生物工学

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物発光光遺伝学2.0:生体内および生体内で光感覚タンパク質を活性化するための生物発光の利用

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

ルシフェラーゼ生物発光レポーターを用いた遺伝子発現のモニタリング細胞自律的な概日時計のリズム

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

細胞毒性の継続とリアルタイム監視のための自律的に生物発光哺乳類細胞

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

生物学的設定における生成と観測化学発光のための新しい技術

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