この作品は、NIH（U54CA143837）、NIH、MDアンダーソンがんセンターサポート助成金（CA016672）、V財団（SAC）、およびKanzius研究財団（SAC、エリー、ペンシルバニア州）からの無制限の研究助成金によって賄われていた。私たちは、行政の支援のために、外科腫瘍科、MDアンダーソンがんセンターのクリスティーン·アッシュに感謝します。

完全な実験の概要を図1に示されている。 さらなる詳細は、以下の手順1〜3に示されている。 1。 NPコロイドの高周波加熱を評価する：例としてAuNPs <ol><li>一般に、各NPサンプルが調査され、第一のバックグラウンドイオン及び汚染物を除去するために脱イオン（DI）水を用いて遠心分離フィルターを通して試料を数回洗浄する。すべてのイオンおよび汚染物質が洗い流されている液体が、DI水と同様の高周波加熱速度（時間）がAuNP懸濁液から削除されています。この精製プロセスはまた、得られるNPのより高い濃度を可能にする。なお、この例ではAuNPsを用いているが、基本的な原理は 、他のNPの材料に適用することができることは注目に値する。 </li><li>一例として、直径5nmの市販のAuNPsの500ミリリットル瓶を浄化した後、電気-Fiは13.56MHzのRFフィールドにそれらをさらすELD強度90 kVの/ M。 <ol><li> 〜6 50kDaの遠心フィルターチューブの間に株式AuNPソリューションとスプリットから125ミリリットルを取る。 3,000 rpmで遠心分離する。 2分5秒。より多くの保存溶液で濾過液リフィルフィルタを削除。すべての500ミリリットルがフィルタリングされるまで繰り返します。 </li><li> DI水の同じような量で濾過液を交換し、約8倍（またはフィルタリングされたバッファのRF時間DI水と同等になるまで）を繰り返します。ノート、UV-Visの分析はまた、汚染物質の吸収ピークを監視するために使用することができる。バッファ汚染物質が各フィルタに、ピペットを約0.5ミリリットルのDI水を完全に除去され、繰り返しピペッティングによりAuNPsを再懸濁したら。これは完全にフィルタからAuNPsを削除し、完全な再懸濁を可能にすべきである。 1 15ミリリットルエッペンドルフチュー​​ブにすべての6懸濁液を兼ね備えています。 </li><li> AuNPsを精製し、濃縮された後、ICP-OES、および/またはICP-MS、UV-VISとconcentratio上のデータのためのゼータ電位を使用してサンプルを分析するnおよびNP安定性であった。 SEMおよび/またはTEM分析は、形態学的データを得るために使用することができる。これらの技術についての詳細なサンプル調製は、文献4に見出すことができる。 </li></ol></li><li> Kanzius高周波これまでの研究8に記載されているシステム、またはこのシステムの導出、（無サンプルプレゼントに）空気中のRF電界がなるように1.3ミリリットルの円筒状の石英キュベットを置く〜キュベット内部の90 kVの/ Mを用い。標準生理食塩水試料（0.9％NaCl）をするための電界は、〜1.1 kVの/ mまで減少するであろう。これらの比較は、異なるシステム間で行われることを可能にするために使用されるおおよその条件である。 <ol><li>ピペット石英キュベットに精製されたAuNPコロイド1,000 mg / Lの試料を1.3ミリリットルとRFフィールドにこれを紹介する。これは、特注のテフロン（登録商標）サンプルホルダーを使用することによって行うことができる。 120秒またはサンプルが電気アークや急激な沸騰を防ぐために、70℃に達するまでの期間、RFフィールドにサンプルを公開しています。カナダ赤外線カメラ·関連ソフトウェアを使用して熱画像データ（および制御領域）pture。この手順を3回繰り返します。 </li><li> DI水バッファからAuNPsを抽出するために別の50 kDaの遠心フィルターを通して試料を濾過する。 3回は再び、RFフィールドにバッファを再公開します。 AuNPコロイドおよび背景DI水バッファ間のHRの差がAuNPs自身のために人事を決定します。 AuNPに〜0.25℃/秒の依存人事を与える〜0.3℃/秒と0.05℃/秒の時間得ることを期待しています。 試験管内/ vivo実験用の1.3ミリリットルの水に、フィルタからの残りAuNPsを再懸濁します。 </li></ol></li></ol> 2。ナノ支援RF誘発性高体温：in vitro試験 <ol><li>これらのインビトロ研究は、2D単層を形成し、癌細胞型の任意のタイプに適用することができる。この実験では、ヒト肝細胞癌は、Hep3B細胞由来の使用。 <ol><li>プレート〜5万CEL増殖培地1mlの12ウェルプレートの前面3つのウェル中のls。 （コントロールとしてのNPの研究と3枚のために3枚を使用してください）​​この6回繰り返します。 NPSを導入する前に24時間、37.5℃でインキュベートする。 5分間のバイオセーフティキャビネット紫外線露光を​​用いて最初にNPSを殺菌する。 </li><li>それぞれにうまく千mg / LのAuNP溶液0.1mlを導入し、さらに24時間のままにしておきます。 3対照細胞プレートの各ウェルに0.1mlの水を加え、さらに24時間のままにしておきます。 </li><li> 24時間後に細胞培地を吸引し、任意の表面に結合したAuNPsを除去してPBSで洗って、合格しています。細胞培地を交換してください。セルは現在、RF被爆の準備ができている。 </li></ol></li><li> RFフィールド内の各12ウェルセルパックを置きます。細胞は31℃に冷却するまで待ちますRF発生器の電源を入れ、3.5分間さらす。細胞培地の最終温度は〜37℃になりますRFフィールドをオフにします。細胞を除去し、分析の前に24時間、インキュベーターに配置します。 <ol> &lt;李&gt;インキュベーターと吸引細胞培地から細胞を取り出します。 MTT試薬の0.4ミリリットルでなく、各ウェルに細胞培地の1.6ミリリットルを追加します。 4時間細胞をインキュベートする。培地を吸引し、ジメチルスルホキシド（DMSO）2mlで置き換える。ベンチロッカーに細胞プレートを配置し、DMSOは、MTT試薬を可溶化することを可能にするために10分間のままにしておきます。最後に、光学的にピペット100 96ウェルプレートにそれぞれの液とは、そのようなSPECTROstarナノプレートリーダーなどのプレートリーダーを用いて570nmでよくお読みください。 </li></ol></li></ol> 3。ナノ支援RF誘発性高体温：in vivo試験 <ol><li>これらのインビボ研究は、同所性または異所性マウスモデルにおいて、固形腫瘍を形成する任意の種類の癌にも適用することができる。この実験は、異所性腫瘍をBALB-CヌードマウスモデルでのHep3B肝癌細胞を使用する。 </li><li>注：すべてのin vivo実験は、関連するすべてのガイドライン、規制、規制当局に準拠して実行されます。また、実証されているプロトコルは、テキサス大学MDアンダーソンがんセンターの施設内動物管理使用委員会（IACUC）の指導と承認の下で行われた。 <ol><li>適切な増殖培地で、組織培養フラスコ中で適切な数の細胞（〜100 k）を成長させる。細胞培養の間、5％CO 2、37℃インキュベーターでインキュベートする。 </li><li>トリプシンで細胞を処理（フラスコから分離するために）、すべての25μlのための200万個の細胞の溶液を生成します。 （氷上）マトリゲル等量を追加し、最終的な注射液を調製し、十分に混合。マウスの背中に所望の位置に、このソリューションを注入し、腫瘍が目的のサイズ（ほとんどの細胞のための2-4週）に成長するために適切な時間を待つ。 RF被曝の前に、BALB-cヌードマウスは、固形腫瘍が子宮外に0.5〜1センチメートル直径を負担する必要があります。 </li></ol></li><li><ol><li> ANAEことにより（この場合、BALB-Cヌードマウス）が使用されたマウスを作製IP注射によるケタミンおよびキシラジンの溶液でそれらをsthetizing。マウスは眠って下落している一方で、37℃に温度制御された室内に保管してください全部で20匹のマウスは、全て同様のサイズ軸受腫瘍を必要とされるであろう。無AuNP注射で両グループ：残りの10匹は、RF-曝露と非RF-曝露のコントロールの間で分割され、一方10匹のマウスを（後者は、PBS注射のみ）と一緒にしてAuNPsせずに使用されます。 </li><li>いったん適切に麻酔、27 Gの針を1-CCの注射器を使用して、腫瘍に直接AuNPsを注入する。 AuNP液をPBSの0.1ミリリットルで200 mg / LでのAuの濃度である必要があります。注射後、血液を吸収し、拭き、アルコールで注射部位を拭くために、外科綿棒を使用しています。 </li><li>次いで、RF発生器の受容頭部に処理されるべきマウスを取り付ける。腫瘍が送信ヘッドに最も近くなるようにマウスを配置しなければならない。処置されるべきされていない領域を遮蔽、ならびに感度銅テープと、目、耳、手足などの分野をTIVE。電荷の蓄積が発生しないように銅テープを適切に接地面と接触していることを確認してください。また、露光領域は、所望の治療部位の大きさよりも少なくとも1cmのギャップを持っている必要があります。 </li><li>位置赤外線熱カメラ、腫瘍および治療領域が表示されるように。 5分間のRFをオンにします。生じた温度曲線を記録します。治療は、直ちに温度を超える42℃の停止処理を実現した場合。 </li></ol></li><li><ol><li>彼らが意識的になるまでのRF曝露後に暖かい室内での麻酔からマウスを回復する。 </li><li> （ - 3.4.1 3.3.1ステップ）48時間後に同じマウスで実験を繰り返す。 </li><li>実験後、制度的なプロトコルと手順に従ってマウスを安楽死させる。腫瘍の重量を記録します。組織学的分析のためにホルマリンを使用して腫瘍を固定し、パラフィンに埋め込む。腫瘍切片は通常、ウィットに染色される例えば Ki-67の時間ヘマトキシリン-および治療 ​​の有効性を測るに関連するターゲットについては、カスパーゼ3 などを切断した。 </li></ol></li></ol>

<ol>
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C. 116, 24380 (2012).</li><li>Kruse, D. E., 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+Radio-Frequency+Coupling+Network+for+Heating+of+Citrate-Coated+Gold+Nanoparticles+for+Cancer+Therapy:+Design+and+Analysis.">A Radio-Frequency Coupling Network for Heating of Citrate-Coated Gold Nanoparticles for Cancer Therapy: Design and Analysis.</a> IEEE Transactions on Biomedical Engineering. 58, 10 (2011).</li><li>Li, 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=Negligible+absorption+of+radiofrequency+radiation+by+colloidal+gold+nanoparticles.">Negligible absorption of radiofrequency radiation by colloidal gold nanoparticles.</a> J. Colloid Interf. Sci. 358, 47 (2011).</li><li>Liu, X., Chen, H. J., Chen, X., Parini, C., Wen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+frequency+heating+of+gold+nanoparticle+dispersions+for+non-invasive+thermal+therapies.">Low frequency heating of gold nanoparticle dispersions for non-invasive thermal therapies.</a> Nanoscale. , (2012).</li><li>Sassaroli, E., Li, K. C. P., O'Neill, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio+frequency+absorption+in+gold+nanoparticle+suspensions:+a+phenomenological+study.">Radio frequency absorption in gold nanoparticle suspensions: a phenomenological study.</a> J. Phys. D App. Phys. 45, 075303 (2012).</li><li>Worle-Knirsch, J. M., Pulskamp, K., Krug, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oops+they+did+it+again!+Carbon+nanotubes+hoax+scientists+in+viability+assays.">Oops they did it again! Carbon nanotubes hoax scientists in viability assays.</a> Nano Lett. 6, 1261 (2006).</li></ol>

我々は、開示することは何もありません。

これらのプロトコルは、実験者が完全に（この場合AuNPs）は、癌治療のためのRF誘導性高体温を増加させることができるためにナノ物質程度を分析することを可能にする。第一のプロトコルは、特に高濃度精製AuNPサンプルから熱産生を分析して扱っています。他のグループは、主にAuNPsがAuNPs自身9-11にせずに懸濁させるバッファから熱産生を報告しているが、それらのRFシステムは、?...

（それらの固有の電気誘電率）の生体組織によるRFエネルギーの吸収は、最終的には温熱により細胞死をもたらす時間の関数として高められた組織温度をもたらす。それは、癌温熱療法は、癌細胞内に内在化し、隣接する健康な無傷の、正常細胞を残して、RF熱変換体として作用する標的化ナノ材料の使用によって最適化することができると仮定される。いくつかの報告が既にNPの様々な癌壊死1-4助剤として有効なRF熱源として作用することが示されている。 両方のインビトロおよびインビボ RF実験に使用した場合にこれらの点は、金のNP（AuNPs）3-5、カーボンナノチューブ1、及び量子ドット6において、図7は、刺激的な特性を示している。 RF場に露出これらのNPの加熱機構の正確な性質は、依然として、一連の議論されているがAuNPsを使用した基本的な実験は、NPのサイズと集約状態の両方で大きな意義を置いている。これは、RF場8にさらされたときの直径を有するのみAuNPs &lt;10ナノメートルで加熱することが示された。 AuNPsが凝集している場合にも、この加熱機構を大幅に減衰する。この集約状態は、効果的なRF療法4用endolysomal細胞内コンパートメント内AuNPコロイド安定性を最適化する際に重要視しin vitroモデル内で検証されました。ただし、このデータを収集し、評価するために使用される技術及び実験的な原理は、特にNPコロイドからRF熱プロファイルを検証する場合には、問題となる可能性がある。 いくつかの報告のNPが懸濁されているバックグラウンドイオン性懸濁液のジュール加熱は、主RFの熱発生源ではなく、それ自体のNP 9-12であり得ることを示している。私たちの最近の論文8は、Tを検証したが彼は10ナノメートル未満の直径のAuNPsからの熱を生成する際に、RF相互作用の使用は、この記事の中で、より詳細にこれらのプロトコルを記述することを目指しています。 また、in vitroおよび肝臓癌モデルのin vivo実験の両方において高体温の熱AuNPs薬としての有効性を評価するために必要なプロトコルおよび技術を実証する。我々は、クエン酸をかぶったAuNPsの単純なコロイドに主に焦点を当てているが、同技術は、抗体と化学療法 - 共役複合体のような他のAuNPハイブリッドに適用することができます。これらの原則に付着していることで実験者がうまくいけば、急速に効果的なRF誘導感熱温熱剤であることがあらゆるナノ材料の可能性を評価することができるはずです。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>500 ml gold nanoparticles (5 nm)</td>
			<td>Ted Pella, INC</td>
			<td>15702-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)</td>
			<td>Millipore</td>
			<td>UFC805024/UFC910096</td>
			<td>(4 ml and 15 ml volumes)</td>
		</tr>
		<tr>
			<td>MEM X1 Cell Culture Media</td>
			<td>Cellgro</td>
			<td>10-101-CV</td>
			<td>(add extra nutrients as necessary)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper Tape</td>
			<td>Ted Pella</td>
			<td>16072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Kanzius RF System (13.56 MHZ)</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR Camera</td>
			<td>FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)</td>
			<td>Contact FLIR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.3 ml Quartz Cuvette</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon Sample holder with Rotary Stage</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPECTROstar Nano Microplate reader</td>
			<td>BGM Labtech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Applied Nanofluorescence, Houston, TX)</td>
			<td>NS1 NanoSpectralyzer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ICP-OES</td>
			<td>PerkinElmer</td>
			<td>Optima 4300 DV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern</td>
			<td>Zen 3600 Zetasizer</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

protocols for assessing radiofrequency interactions with gold nanoparticles and biological systems for non-invasive hyperthermia cancer therapy

既存の対応物よりも毒性および侵襲性癌療法が非常に望ましい。最小限の毒性を引き起こす、身体深部に浸透RF電界フィールドの使用は、現在、非侵襲性癌療法の実行可能な手段として研究されている。これは、内在化ナノ粒子（NPを）有するRFエネルギーの相互作用は、最終的には細胞壊死で終わる、セルの（温熱療法）を引き起こす可能性が過熱する熱を放出することが想定される。 非生物学的システムの場合、我々は、高濃度のNPコロイドによって遊離熱を定量化に関する詳細なプロトコルを提示する。生物系は、in vitro実験の場合には、我々は、効果的に、バルク媒体の加熱アーチファクトが有意にデータを不明瞭にすることなく、RFエネルギーに癌細胞を露出させるために接着されなければならない技術および条件を記載している。最後に、我々は詳細な方法論Fを与えるまたは異所性肝癌腫瘍を用いたin vivoマウスモデルで 。

1。例としてAuNPs：NPコロイドのRF加熱を評価する。 セクション1.1に従った後 - 1.2.3を5 nmおよび10 nmの直径AuNPsの高濃縮、安定し、かつ精製されたソリューションを持っていることを期待しています。として購入したストック溶液を500ミリリットルから1,000ミリグラム/ Lの濃度で溶液の少なくとも4ミリリットルを得ることを期待図2に示されているようにAuNPsこの?...

Watch this Scientific Journal Video about Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy at JoVE.com

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy | Medicine | Video

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

私たちは、13.56MHzの高周波（RF）の両方の非生物学的および生物学的システムにおける金ナノ粒子コロイドと電気分野（ 試験管内 / 生体内 ）の相互作用を調査するために使用されるプロトコルについて説明します。これらの相互作用は、癌治療における用途のために研究されている。

非侵襲ハイパーサーミアがん治療のための金ナノ粒子と生体系とラジオ波の相互作用を評価するためのプロトコル

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

protocols-for-assessing-radiofrequency-interactions-with-gold

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10.7K ビュー.  University of Texas M.D. Anderson Cancer Center. 私たちは、13.56MHzの高周波（RF）の両方の非生物学的および生物学的システムにおける金ナノ粒子コロイドと電気分野（ 試験管内 / 生体内 ）の相互作用を調査するために使用されるプロトコルについて説明します。これらの相互作用は、癌治療における用途のために研究されている。 

ビデオ: Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

ヒト膵島の染色プロトコル

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

医学

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

がん治療のモニタリングのためのチェレンコフ発光イメージング（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

乳幼児の脳代謝と血行動態の非侵襲的光計測

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

微小血管と内皮機能の非侵襲的評価

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

縦用の眼の前房に移植、非侵襲<emインビボ&gt;</emリアルタイムでのシングルセル分解能&gt;イメージング

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

と陽電子放射断層撮影法を用いた非侵襲的イメージングとリビングラットにおける脳虚血の分析<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

卵巣がん再発を検出し、モニターするための非侵襲的イメージングのためのマウスモデル

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

生きたマウスのシリアル内視鏡イメージングを使用して、腸の異常のための新しい治療の有効性の非侵襲的評価

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

マウスで内がえし筋機能の非侵襲的評価

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.