我々は、生体工学およびミズーリ大学のクリストファーS.ボンド生命科学センターの省の支援を認める。私たちは、ミズーリ州のライフサイエンス研究会09〜1034とNIH R21CA139186 - 0からの助成金を認める。私たちは、ミズーリ大学、ミズーリ分子細胞学コアの大学、および財政的支援のための技術のミズーリ大学大学ライフサイエンス学部研究機会プログラムをも感謝。

1。血液サンプル調製<ol><li> 2つの5 mLのvacutainersにIV期のがん患者から全血の約10 mLを描く。 </li><li>毎分3000回転（rpm）で10分間チューブを遠心する。 </li><li>血液サンプルは、3つの異なる層に分離されます。下部層は、また、 バフィコートと呼ばれる赤血球、中間層を含み、白血球およびメラノーマ細胞が含まれており、最上層は血漿および血小板が含まれています。バフィコートを乱すことなく、できるだけ多くのプラズマとして削除します。 </li><li> 2つの1 mLのウイントローブ管のHistopaque 1077の代わりに250μL。 </li><li> 9&quot;パスツールピペットを使用して、&lt;500μLを収集することを確認しながら、残りの血漿、全体バフィコート、さらに赤血球の最上層を抽出する。 </li><li> Histopaque層の上にソリューションを配置し、3000rpmで10分間、15 mLコニカルチューブにウイントローブ管を遠心する。 </li><li>血液は再びれます独立したが、今回は、赤血球は、バフィコートの簡単な抽出を可能にする、Histopaqueで区切られます。全体のバフィコートを取得し、2 mLのエッペンドルフチュー​​ブに配置する9&quot;パスツールピペットを使用してください。 </li><li> 2パーセント/ VのTween20を含むPBS 1.5 mlのバフィーコートを一時停止する。 </li></ol> 2。フローチャンバーの建設<ol><li>アクリルシリンダー、20ミリメートル、外径17内径、および高さ8ミリメートルを取得します。互いに90度の角度で円筒の側面を介して3つの5 mm径の穴を開けます。ポリマーチューブの3枚（1.6内径5 mmの外径）を切断し、ドリル穴に入れます。 </li><li>水タイトなシールを作るためにアクリルリングの下部にパラフィルムを伸ばす。パラフィルムは、アクリルリングと浅いボウルを形成し、フローチャンバーを形成するアクリルアミド溶液を開催します。 </li><li>の向かい側にあるチューブの部分を段階的に1.6 mmの直径のワイヤーを一時停止お互いは、単独で配線がリングの真ん中から見えるが認識するように。 1.6mmの直径のワイヤーと位置離れて一時停止線からチューブ1 mmのチューブの第三のピースを埋める。これらのワイヤは、チューブに入ることからアクリルアミドを防ぎます。アクリルアミドが固化した後、ワイヤーが光ファイバを配置する円筒状の流路と場所を残して、抽出される流路に焦点を当てています。 </li><li>小さなビーカーに準備アクリルアミドを5 mL [20 gのアクリルアミド（シグマアルドリッチ）、0.7 gのビスアクリルアミド（シグマアルドリッチ）、蒸留水100mL]を追加します。過硫酸アンモニウム（シグマ）の0.02グラムを測定し、ビーカーに追加します。 3分間撹拌棒で混ぜる。ビーカーに20μLtetramethylethylenediamene（フィッシャーサイエンティフィック）を追加します。すぐにトップに充填、アクリルリングに混合物を注ぐ。数秒後、溶液、ゲル、フローチャンバーの金型を作っています。慎重にアクリルアミドゲルを邪魔しないことを確認して、ワイヤーを引き出します。フローチャンバBERが使用できるようになりました。 </li></ol> 3。光音響流量計のセットアップ<ol><li>音響的にカップルに直接流路の上に超音波ゲルを使用してフローチャンバーへのポリフッ化ビニリデン圧電音響トランスデューサ（パナメトリクス）。 </li><li>バヨネットニール- Concelman（BNC）ケーブルを使用して、RITECブロードバンドレシーバ- 640Aの入力（：外、ハイパスフィルタ：1 MHzで、入力強度インピーダンス：50オーム、増幅：35デシベルローパスフィルター）への変換器を接続する。オシロスコープ（テクトロニクスTDS 2024B）にフィルタの出力を接続してください。オシロスコープをトリガするために、フォトダイオード（Thorlabsは）変換器の近くに設定されており、BNCケーブルを使用してオシロスコープに接続されている。 </li><li> T -コネクタ（スモールパーツ）はフローチャンバーの一方のアームに取り付けられています。二つのシリンジポンプ（ブレーントリーサイエンティフィック）は、コネクタの他の支店に接続されています。他のパート1で調製した細胞懸濁液を含むのに対して、一つのシリンジは、空気を含んでいます。に流量を設定する100μL/分。 </li><li>エアフォームの小さなポケットまで、フローチャンバーの小さいチューブを抽出し、そのスロットの0.37の開口数と直径1mmのコア光ファイバを挿入する。インタフェース信号を削減し、流路に適用されるレーザー光の量を増やすために水でポケットをいっぱいに。流路に光ファイバと短い距離の開口数のために、レーザーは、直接レーザーの目の前のサンプルでは、​​光音響信号を作成する場合は、サンプルであることを保証する、任意の時点でサンプルの一つスラグを照射します。レーザーのエネルギーは7 mm 2のスポットサイズで約28ミリジュール/ cm 2とする必要があります。 </li><li>シリンジポンプの両方をオンにします。 2つの不混和流体は、T字路に到達すると、彼らが均質ナメクジとトランスデューサ過去のフローに分割されます。 </li><li>ナノ秒メラノーマ細胞における光音響波を生成するためにレーザーをパルスで流れる試料を照射する。メラニンはbroadbであるとしてと吸収光学、目に見えるレーザーの波長を使用することができます。 </li><li>スラグは、トランスデューサーの上になったとき、信号がオシロスコープに表示される場合は、スラグには、黒色腫が含まれており、コレクションバイアルにドロップするまで、システムを介して追跡する必要があります。 </li></ol> 4。免疫細胞化学<ol><li>スライドガラスにそれらを修正するためにサイトスピン4セントリフュージ（サーモサイエンティフィック）にキャプチャしたナメクジを取る。場所のガラスはCytofunnels（ブラウンフィルターカードサーモサイエンティフィック社とシャンドンEZシングルCytofunnel）に挿入し、サイトスピン4遠心分離機にそれらをロードする。各漏斗にPBSで（シグマ）1％ウシ血清アルブミンの100μLを入れ、次に捕捉細胞はスライドガラスに付着するための3分間600rpmで遠心する。 </li><li>遠心分離後、Cytofunnelsに細胞サンプルを追加し、3分間600rpmで遠心する。その後、エタノールベースの​​固定液とスライドをスプレー。 </li><li>二回10分間PBSでスライドを洗浄します。 </li><l私は&gt;非特異的結合をブロックするには、ヤギ血清（Sigma社）と室温で1時間PBSの90μLの10μLの混合物で各スライドをインキュベートする。 </li><li>インキュベーション後、スライドをデカントし、室温で加湿チャンバー内で1時間一次抗体溶液でインキュベートする。各スライドには、1％ウサギで提起されたMART - 1抗体、1％マウスで育ったCD - 45抗体、10％ヤギ血清、89％PBSを含める必要があります。 </li><li>インキュベーション後、10分を3回PBSでスライドを洗う。 </li><li>スライドはその後、蛍光体抗体に結合した二次抗体溶液でインキュベートされる。混合物は0.05％ヤギ抗ウサギ抗体、0.05％のヤギ抗マウス抗体、0.1％ヤギ血清、99％PBSを含んでいます。スライドを室温で1時間、暗所でインキュベートする。 </li><li>インキュベーション後、スライドを5分間PBSで3回洗浄デカントする。 </li><li>核のために染色する、のためにDAPI 0.1μg/ mLの（インビトロジェン社）で細胞をインキュベート1分。その後、スライドをデカントし、PBSで洗浄する。 </li><li>スライドごとの樹脂ベース取付メディアの20μLを用いて細胞サンプル上にカバースリップをマウントします。スライドを維持するために明確なマニキュアでカバースリップをシール。今すぐスライドは蛍光顕微鏡で撮像される準備が整いました。 </li></ol> 5。代表的な結果： <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="図1"/> 図1白血球やメラノーマ細胞からの光音響波形がここに表示されます。白血球は、ない固有の色素沈着のない、電子ノイズのフラットライン（左）とは光音響波とマニフェストを生成しません。色素性メラノーマ細胞は、強力な光音響波を（右）が生成されます。 <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="図2"/> 図2：免疫細胞化学染色した後、培養メラノーマ細胞はBLにDAPIシグナルを示すMART1一方、UEは、緑色で強調表示されます。 <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="図3"/> 図3。白血球の指標としてCD45と、図2に、DAPIとMART1用に画像をオーバーレイ、この図は、いくつかの白血球細胞の間でメラノーマ細胞を示しています。

<ol>
	<li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li><li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li><li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li><li>Weight, R. M., Dale, P. S., Viator, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+melanoma+cells+in+human+blood+using+photoacoustic+flowmetry.">Detection of circulating melanoma cells in human blood using photoacoustic flowmetry.</a> , 106-109 (1985).</li><li>Weight, R. M., Dale, P. S., Lisle, A., Caldwell, C., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+detection+of+metastatic+melanoma+cells+in+the+human+circulatory+system.">Photoacoustic detection of metastatic melanoma cells in the human circulatory system.</a> Opt. Lett. 31, 2998-3000 (2006).</li><li>Gupta, A., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geometry+on+droplet+formation+in+the+squeezing+regime+in+a+microfluidic+T-junction.">Effect of geometry on droplet formation in the squeezing regime in a microfluidic T-junction.</a> Microfluidics and Nanofluidics. 8, 799-812 (2010).</li><li>Mocellin, S., Hoon, D., Ambrosi, A., Nitti, D., Rossi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+circulating+tumor+cells+in+patients+with+melanoma:+a+systematic+review+and+meta-analysis.">The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis.</a> Clinical Cancer Research. 12, 4605-4605 (2006).</li><li>Cristofanilli, M., Budd, G., Ellis, M., Stopeck, A., Matera, J., Miller, M., Reuben, J., Doyle, G., Allard, W., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> New England Journal of Medicine. 351, 781-781 (2004).</li><li>Cristofanilli, M., Hayes, D., Budd, G., Ellis, M., Stopeck, A., Reuben, J., Doyle, G., Matera, J., Allard, W., Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+a+novel+prognostic+factor+for+newly+diagnosed+metastatic+breast+cancer.">Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer.</a> Journal of Clinical Oncology. 23, 1420-1420 (2005).</li><li>Ghossein, R., Scher, H., Gerald, W., Kelly, W., Curley, T., Amsterdam, A., Zhang, Z., Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+cells+in+patients+with+localized+and+metastatic+prostatic+carcinoma:+clinical+implications.">Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.</a> Journal of Clinical Oncology. 13, 1195-1195 (1995).</li><li>Loberg, R., Fridman, Y., Pienta, B., Keller, E., McCauley, L., Taichman, R., Pienta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+isolation+of+circulating+tumor+cells+in+urologic+cancers:+a+review.">Detection and isolation of circulating tumor cells in urologic cancers: a review.</a> Neoplasia. 6, 302-302 (2004).</li><li>Viator, J. A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance.">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li></ol>

ジョンA.ビアトールは、創設者とビアトールテクノロジーズ社、人間の健康の改善のための音響技術を商業化するために形成された会社の社長です。

CTCを検出することはまれな腫瘍細胞を単離するのが難しい問題があるため、非常に少数の臨床応用でも、研究集約的な分野です。他の多くの技術がRT - PCR、マイクロセルのキャプチャ、免疫キャプチャ、および他の方法8月12日を含めて、CTCの検出、のために評価されている。しかし、CMCのショーの光音響検出は、ラベルフリーで、小さな、光吸収粒子を捕捉するために迅速かつ正確な手段を提供するように約束。 白血球を分離するために記述されたプロトコルは、非常に効率的です、まだ赤血球の数値が低いほど、サンプルに存在している。これらルージュ混入細胞はまた、レーザー光を吸収するが、大幅に減少したレベル。赤血球は、当社の黒色腫の検出システムに干渉しないように、五健康な患者のサンプルは、システムを介して導入され、いずれも赤血球が十分に低い濃度で存在していることを示す、シグナルを明らかにしていない無視される。 濃度の一連の研究は、培養メラノーマ細胞を用いて行われていると光音響流のシステムがダウンして1セル/μL（未発表データ）に敏感な証明されています。これらの研究は継続にあり、すぐに癌の患者のサンプルを用いて試験が含まれます。 この光音響法はまた、外因性発色団を取り付けることで、乳癌や前立腺癌などの非色素CTCの、で使用するために評価されている。我々は癌cells13に金ナノ粒子を用いた予備作業を実行しました。これらのテストは、ナノ粒子は、光音響波を生成するために必要な光吸収を提供できることが示されている。残りの技術的課題は、選択的にがん細胞にこのような粒子を添付することです。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> ABCD1234 </td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

循環腫瘍細胞（CTCの）は巨視的な腫瘍から分離し、種子の二次腫瘍1,2,3への血液やリンパ系を介して拡散しているそれらの細胞である。 CTCのは、転移性疾患の指標であると血液サンプル中のそれらの検出は、癌の診断と治療に対する患者の反応を監視するために使用されることがあります。 CTCのは稀なので、進行がん患者、それらの検出と列挙内の正常な血液細胞の十億の中の腫瘍細胞について、構成することは困難な作業です。我々は4,5メラノーマ細胞（CMCの）循環を検出するためのカスタムフローサイトメーターでの超音波を誘導音響生成する最もメラノーマ細胞における色 ​​素の存在を利用する、またはレーザー。このプロセスは、遠心分離を使用し、白血球の層を得る全血検体を分離伴います。全血中に存在する場合、CMCは同じような密度のために白血球を分離します。これらの細胞を再懸濁するリン酸緩衝生理食塩水（PBS）と流量計に導入する。むしろ血液細胞懸濁液の連続的な流れよりも、我々は、さらなる研究のためにこれらの細胞を捕捉するために、二相流を誘導した。二相流では、マイクロ流体システムに2つの不混和液体は液体6,7のナメクジを交互に接合し、フォームに会う。 PBSは、順番にレーザー光を照射している白血球や空気のフォームマイクロリットルのナメクジを中断。液相に界面活性剤の添加は、均一なスラグ形成を可能にし、ユーザーは、2つのフェーズの流量を変化させることによって、異なるサイズのナメクジを作成することができます。白血球と空気とPBSのナメクジのナメクジは光吸収剤を含まず、従って、光音響波を生成しない。しかしながら、単一のCMCを含む白血球のナメクジは、レーザー光を吸収し、高い周波数の音波を生成する。光音響波を生成するこれらのナメクジはverificのために隔離し、細胞化学染色のために収集されますCMCを注意深く検討する必要があります。

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

我々は転移性疾患の早期指標として循環するメラノーマ細胞を検出するためにレーザー誘起超音波を用いてフローサイトメーターを開発しました。

光音響流量測定法を用いて循環メラノーマ細胞の検出と分離

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

15.7K ビュー.  University of Missouri. 我々は転移性疾患の早期指標として循環するメラノーマ細胞を検出するためにレーザー誘起超音波を用いてフローサイトメーターを開発しました。

ビデオ: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

患者の血液サンプルから生存循環腫瘍細胞の急速な分離

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

電気化学センサを用いた細菌検出＆識別

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

統合された光音響眼底検査とスペクトラルドメイン光コヒーレンストモグラフィー

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光音響膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

マイクロトロイドの光共振器を用いたラベルフリー単一分子検出

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

ゴールドナノロッドを含む細胞車の作製と光音響分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

DNA結合色素化学と液滴デジタルPCRを使用したマイクロRNA定量循環

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

切り替え可能な音響光学分解能光音響顕微鏡<em&gt;インビボ</em&gt;小動物の血管系イメージング

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...