我々はOR人材の育成と計画のための夫人伊那Wesselman恩義があります。

1。術中マルチスペクトル蛍光カメラ<ol><li>カスタムメイドのカメラシステムは、SurgOptix（SurgOptix株式会社、レッドウッドショアーズ、カリフォルニア州、米国）と共同で緊密に生物医学イメージングのための研究所（IBMI、工科大学/ヘルムホルツZentrum、ミュンヘン、ドイツ）で開発さで開発されました。術中マルチスペクトル蛍光カメラシステムのセットアップを図2に示されています。 </li><li>光は、光学系のシステムを通過し、可視光、発光（蛍光）の波長帯における光と励起波長帯の光に分離される。これらのバンドルは、CCD -カメラ（CCD）（図2）によって検出されています。すべてのカメラからのマルチスペクトル信号は、アーチファクトを補正し、真の定量的な蛍光色素の生体分布を得るために処理されます。色と蛍光信号は、外部モニタ上で個別の画像として表示されるか、一つの画像に重ね合わせることができます。 </li><li>カメラの励起と発光のフィルターはそれぞれ750 nmのおよび800 ± 20 nmで収集する測定ここで設定されます。 </li></ol> 2。光造影剤の調製<ol><li>蛍光造影剤は、グリーン（ICG、Pulsion AG、ミュンヘン、ドイツ）は無菌条件下で調製されるインドシアニン。は0.5 mg / mLの濃度が使用されます。後者は集計にICGを発生するので、滅菌蒸留水ではなく、塩化ナトリウム（NaCl）を使用することが重要です。 </li><li>調製後、溶液を漂白することにより、蛍光強度の速い劣化を避けるために、暗い、涼しい場所に保管する必要があります。注：このポイントから前方に光から保護ICGを保つ。 </li></ol> 3。術中イメージング - ICGのセットアップと注入<ol><li>カメラが配置され、標準的な外科手術との干渉を最小限に抑えるために手術前に手術室で開始され、高精細画面（図2B）に接続されている。 </li><li>カメラは、滅菌や衣服の研究者によって標準的な滅菌ドレープ（カールツァイスビジョンBV、スリードレヒト、オランダ、OPMIドレープREF 306071）で覆われている。 </li><li>注：色付きの消毒剤はしばしば自家蛍光であるとイメージングの手順に干渉する可能性があるとして、唯一の明確な消毒剤を使用するように注意を払わない。また、滅菌ドレープと手術のマーカーは、自家蛍光することができます。それは手術前に自家蛍光のためにORと営業分野で使用されるすべての材料をテストすることをお勧めします。 </li><li>蛍光造影剤は、グリーン（ICG）は無菌条件下で調製されるインドシアニン。は0.5 mg / mLの濃度が使用されます。後者は集計にICGの原因となるので、それは、NaClの滅菌蒸留水を使用することが重要とではありません。調製後、溶液を蛍光強度の速い劣化を避けるために、暗い、涼しい場所に保管する必要があります。注：それは、ICGを準備した後、手袋を変更することをお勧めしますICGの流出のにも少量のように画質に影響を与える可能性があります。 </li><li>撮像法は、腹部を開いた後に開始されます。関心の領域が露出されると、カメラが動作してフィールドに操縦されている。ズームとフォーカスは、無菌条件下で調整されます。手術室の灯りは、蛍光信号の良好な検出のためにオフになります。 </li><li>は0.5 mg / mLのICGの1.0 mLを、1つの注射器で標準青色色素（パテントブルー/ブルーpatenté、ゲルベ、フランス）の1.0 mLを混合される。外科医は、エージェントの流出を防止する、原発腫瘍周囲4つの象限に造影剤を注入します。注：手術用手袋でICGの流出の場合には、それが手袋を変更する必要がある。静止画像やリアルタイムのビデオは、リンパの流れと蛍光センチネルリンパ節（図3）の外観を取得しています。すべての画像やビデオを直接コンピュータに保存されます。 </li><li>離れて蛍光から、SLNは、どちらガンマプローブまたは目視検査によって青色変色するため、または両方を使用して、標準プロトコルにしたがって検出される。放射性トレーサーは、一般的にlymphoscintigramが放射性SLNの検出のために実行された後、手術前に一日に投与される。これは標準的な手順の一部であり、蛍光イメージングプロトコルで場所がない。 </li><li> SLNのリアルタイム切除は、リンパ節の蛍光と青色の変色の両方によって導かれる。カメラのリアルタイム機能は、任意の残存蛍光または蛍光灯のノードの予想外のローカライズのために検出するのに役立ちます。 </li><li> SLNの切除後に、ex vivoでの画像は、蛍光シグナルの存在を全摘出したリンパ節の取得されています。 （図4）静止画をキャプチャするときに露光時間は、より高い分解能のために延長することができます。 </li><li>リンパ節の病理組織学的検査ではセンチネルリンパ節の腫瘍細胞の有無を明らかにする。 </li></ol> 4。 Representative結果蛍光リンパ節は、高い信号対バックグラウンド比を検出することができます。また、リンパ管を通っICGの流れをリアルタイムでリンパ節のマッピングを可能にモニタすることができます。結果は、おそらく別の照明の手順は、検出を達成するために必要とする、ノードの深さの増加によって影響を受ける可能性があります。特定の腫瘍標的蛍光剤の将来のアプリケーションは、この技術を用いて癌細胞陽性SLNの術中検出を提供することがあります。 <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="図1" /> 図1。センチネルリンパ節（SLN）の理論。SLNは、腫瘍からの最初の流入領域リンパ節（秒）です。 <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="図2" /> 図2。マルチスペクトル蛍光カメラシステムの概略図の写真（）。手術室でのカメラシステムの基本的なセットアップ（B）。 <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="図3" /> 図3。外陰癌におけるリンパ節のマルチスペクトル蛍光イメージング。 生体内のリンパ節（）のカラー画像。 生体内で同じリンパ節、（B）の蛍光画像。カラー画像（C）に重畳された擬似カラー蛍光画像。 <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="図4" /> 図4。子宮頸がんのリンパ節のex vivoでのマルチスペクトル蛍光イメージング。リンパ節生体外で （）のカラー画像。同じリンパ節、ex vivoで （B）の蛍光画像。カラー画像（C）に重畳された擬似カラー蛍光画像。

<ol>
	<li>Tinga, D. J., Bouma, J., Aalders, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patients+with+squamous+cell+versus+adeno(squamous)+carcinoma+of+the+cervix,+what+factors+determine+the+prognosis.">Patients with squamous cell versus adeno(squamous) carcinoma of the cervix, what factors determine the prognosis.</a> Int J Gynecol Cancer. 2 (2), 83-91 (1992).</li><li>McMahon, C. J., Rofsky, N. M., Pedrosa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+metastases+from+pelvic+tumors:+anatomic+classification,+characterization,+and+staging.">Lymphatic metastases from pelvic tumors: anatomic classification, characterization, and staging.</a> Radiology. 254 (1), 31-46 (2010).</li><li>Zivanovic, O., Khoury-Collado, F., Abu-Rustum, N. R., Gemignani, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+biopsy+in+the+management+of+vulvar+carcinoma,+cervical+cancer,+and+endometrial+cancer.">Sentinel lymph node biopsy in the management of vulvar carcinoma, cervical cancer, and endometrial cancer.</a> Oncologist. 14 (7), 695-705 (2009).</li><li>Fuller, J., Guderian, D., Kohler, C., Schneider, A., Wendt, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+edema+of+the+lower+extremities+after+lymphadenectomy+and+radiotherapy+for+cervical+cancer.">Lymph edema of the lower extremities after lymphadenectomy and radiotherapy for cervical cancer.</a> Strahlenther Onkol. 184 (4), 206-211 (2008).</li><li>Ayhan, A., Celik, H., Dursun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+mapping+and+sentinel+node+biopsy+in+gynecological+cancers:+a+critical+review+of+the+literature.">Lymphatic mapping and sentinel node biopsy in gynecological cancers: a critical review of the literature.</a> World J Surg Oncol. 6, 53-53 (2008).</li><li>Tanis, P. J., Nieweg, O. E., Valdes Olmos, R. A., Rutgers, E. J., Kroon, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+sentinel+node+and+validation+of+the+technique.">History of sentinel node and validation of the technique.</a> Breast Cancer Res. 3 (2), 109-112 (2001).</li><li>Ghobashy, A. E. E. l., Saidi, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sentinel+lymph+node+sampling+in+gynaecological+cancers:+techniques+and+clinical+applications.">Sentinel lymph node sampling in gynaecological cancers: techniques and clinical applications.</a> Eur J Surg Oncol. 35 (7), 675-685 (2009).</li><li>Alford, R., Simpson, H. M., Duberman, J., Hill, G. C., Ogawa, M., Regino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+organic+fluorophores+used+in+molecular+imaging:+literature+review.">Toxicity of organic fluorophores used in molecular imaging: literature review.</a> Mol Imaging. 8 (6), 341-354 (2009).</li><li>Themelis, G., Yoo, J. S., Soh, K. S., Schulz, R., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+intraoperative+fluorescence+imaging+system+using+light-absorption+correction.">Real-time intraoperative fluorescence imaging system using light-absorption correction.</a> J Biomed Opt. 14 (6), 064012-064012 (2009).</li></ol>

VNとGMvDは前の手順にSurgOptix社​​の科学委員会のメンバーになって、研究のプロトコルは、ローカルの治験審査委員会（IRB）によって承認され、患者からインフォームドコンセントが取得されます。

このビデオでは、婦人科腫瘍におけるセンチネルリンパ節（SLN）の術中検出のためのマルチスペクトル術中蛍光イメージング技術の応用を示しています。方法論は、従来のSLNの手順に比べていくつかの利点があります。注射は、手術自体ではなく、特に婦人科癌で、より患者に優しいです、手術前に一日、より麻酔患者との間に行​​われます。さらに、術中イメージングは​​ガイガー出納係を経由して間接的にリンパの流れとその排水パターンSLNに、ではなく、直接の視覚的なフィードバックと外科医が用意されています。リンパ節は骨盤内の深くに位置している子宮頸がん、で、これは検出を向上させることができる。 それにもかかわらず、最大の意義は、対象画像に向かってステップアップのアプローチにあります。対象となる蛍光造影剤は、特に腫瘍細胞に向け腫瘍特異的抗体または基質と融合させることができる。このように、術中蛍光イメージングは​​根本的に腫瘍の手術の現在の慣行を変化させる大きな可能性を秘めている。

A correction was made to <a href="http://www.jove.com/index/details.stp?id=2225">Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons</a>. There was an error with an author's name. The author name was corrected to include a first initial as follows:
K. Tim Buddingh
instead of:
Tim Buddingh

A correction to an authors name has been made for the article Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology.

Erratum: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

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<td>Indocyanine Green (ICG)</td>
<td>&nbsp;</td>
<td>Pulsion Medical Systems AG, Munich, Germany</td>
<td>D-81829</td>
<td>A solution of ICG in distilled water is used and not in NaCl 0.9%.</td>
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<td>Patent blue / Bleu patent&eacute;</td>
<td>&nbsp;</td>
<td>Guerbet, Paris, France</td>
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<td>Sterile water for injection</td>
<td>&nbsp;</td>
<td>B. Braun</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Multispectral fluorescence camera system </td>
<td>&nbsp;</td>
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multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology

ほぼ全ての固形腫瘍の予後は、リンパ節転移の有無によって異なります。1-3外科的治療が最も頻繁に腫瘍の流域面積で完全なリンパ節郭清を伴う腫瘍の根治的切除を組み合わせた。しかし、リンパ節の除去が感染、創傷内訳とリンパ浮腫に起因する罹患率に関連付けられている。代替として4,5、センチネルリンパ節の手順（SLN）は腫瘍からの最初の流入領域リンパ節を検出するために数十年前に開発された6 lymphogenic普及の場合には、SLNは（図1）影響されている最初のリンパ節です。したがって、SLNの転移が含まれていない場合、下流のリンパ節にも腫瘍転移がないことを保証し、削除する必要はないでしょう。 SLNの手順では、多くの腫瘍タイプに対して、乳癌および黒色腫のような、だけでなく、外陰部、子宮頸部のがんの治療の一環である。SLN検出のための7現在の標準的な方法は、手術前にradiocolloid一日の腫瘍周囲注射によってであり、術中と色染料。子宮頸部および外陰癌における手続きの欠点は、複数の患者の増加心理的苦痛につながる陰部に注射、および放射性コロイドの使用である。 マルチスペクトル蛍光イメージングは​​、radiocolloidの注射を必要とせずに手術中に適用できる新たな画像診断法です。術中蛍光イメージングのための、2つのコンポーネントが必要とされています：蛍光剤と術中イメージングのための定量的な光学系を。蛍光体として、我々はグリーン（ICG）をインドシアニン使用している。 ICGは、心機能、脳血流と肝灌流を評価するために何十年も使用されています。8それは安全な薬理学的プロファイルを持つ不活性薬である。約750 nmで励起されたとき、それは800 nm付近の近赤外スペクトルで発光する。カスタムメイドのマルチスペクトル蛍光イメージングカメラシステムが使用されました。9。 このビデオの記事の目的は、子宮頸部および外陰癌患者で術中蛍光イメージング法を用いてSLNの検出を実証することである。蛍光イメージングは​​、radiocolloidと青の色素で構成される、標準的な手順と組み合わせて使用​​されます。将来的には、術中蛍光イメージングは​​、現在のメソッドを置き換え、また、乳癌や悪性黒色腫などの他の適応症に対して容易に譲渡可能であるかもしれない。

Watch this Scientific Journal Video about Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology at JoVE.com

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

蛍光イメージングは​​、外科腫瘍学における画像誘導手術のための有望な革新的なモダリティである。このビデオでは、婦人科oncologicyで展示として、蛍光イメージングを用いたセンチネルリンパ節の検出のための技術的な手順を説明します。蛍光剤と一緒にマルチスペクトル蛍光カメラシステム、インドシアニングリーン、適用されます。

婦人科腫瘍におけるセンチネルリンパ節の術中検出のためのマルチスペクトルのリアルタイム蛍光イメージング

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

multispectral-real-time-fluorescence-imaging-for-intraoperative

Research

JoVE Journal

Medicine

23.0K ビュー.  University Medical Center Groningen. 蛍光イメージングは​​、外科腫瘍学における画像誘導手術のための有望な革新的なモダリティである。このビデオでは、婦人科oncologicyで展示として、蛍光イメージングを用いたセンチネルリンパ節の検出のための技術的な手順を説明します。蛍光剤と一緒にマルチスペクトル蛍光カメラシステム、インドシアニングリーン、適用されます。

ビデオ: Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

医学

Stereotactic Radiosurgery for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3 Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4 patient mouthpiece active breathing coordination,5 respiration-correlated computed tomography,6 or image-guided tracking of fiducials implanted within and around a moving tumor.7-9 The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10 Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8 Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET).8 Use of 18F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.

婦人科癌に対する定位放射線

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

微妙な子宮内膜症の術中検出：腹膜整合性の喪失に伴う骨盤痛の検出および治療のための新たなパラダイム

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

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. ...

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 ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

ヒト腹腔器官培養における卵巣癌普及の初期段階のモデル化

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.