OR personelin eğitim ve planlama için Bayan Ina Wesselman&#39;ın borçlu.

1. İntraoperatif Multispektral Floresan Kamera <ol><li> SurgOptix (SurgOptix Inc, Redwood Shores, CA, ABD) ile işbirliği içinde yakın Biyolojik ve Tıbbi Görüntüleme Enstitüsü (IBMI, Teknik Üniversitesi / Helmholtz Zentrum, Münih, Almanya) geliştirilen bir ölçüye göre kamera sistemi geliştirilmiştir. Intraoperatif multispektral floresan kamera sistemi kurulumu, Şekil 2&#39;de gösterilmiştir. </li><li> Işık optik bir sistem geçer ve görünür ışık emisyonu (floresans) dalga boyu bandındaki ışık ve dalgaboyu bandında ışık ayrılır. Bu demetleri CCD kameralar (CCD) (Şekil 2) ile tespit edilir. Çoklu-spektral Tüm kameralardan gelen sinyalleri eserler için doğru ve gerçek kantitatif florokrom biyo-dağıtım verim için işlenir. Renk ve floresan sinyalleri harici monitör ayrı görüntü olarak görüntülenen veya bir görüntü eklenmiş olabilir. </li><li> Kameranın uyarma ve emisyon filtreleri sırasıyla 750 nm ve 800 ± 20 nm toplamak için ölçüm burada ayarlanır. </li></ol> 2. Optik Kontrast Ajan Hazırlık <ol><li> Floresan kontrast madde yeşil (ICG, Pulsion AG, Münih, Almanya) steril koşullar altında hazırlanan indosiyanin. 0.5 mg / ml konsantrasyon kullanılır. Ikincisi agrega ICG neden olarak, steril distile su ve sodyum klorür (NaCl) kullanmak önemlidir. </li><li> Hazırlanmasından sonra, çözüm floresan beyazlatma tarafından hızlı bozulmasını önlemek için, karanlık, serin bir yerde saklanmalıdır. Not: ışıktan korumalı ICG bu noktadan ileriye doğru tutun. </li></ol> 3. İntraoperatif Görüntüleme - Kurulum ve ICG enjeksiyonu <ol><li> Kamera yerleştirilmiş ve standart cerrahi prosedür ile etkileşimi en aza indirmek için cerrahi öncesi ameliyathanede yılında başlatılan, ve yüksek çözünürlüklü ekranlar (Şekil 2B) bağlı. </li><li> Kamera (Carl Zeiss Vision BV, Sliedrecht, Hollanda, OPMI Örtüsü REF 306.071) steril VEYA giyim araştırmacı tarafından standart, steril örtüler kaplıdır. </li><li> Not: renkli dezenfekte maddeleri genellikle autofluorescent ve görüntüleme prosedürü ile etkileşebilir, sadece açık dezenfekte maddeleri kullanmaya özen. Ayrıca, steril örtüler ve cerrahi belirteçlerinin autofluorescent olabilir. Bu cerrahi öncesi otofloresans OR ve işletme alanında kullanılan tüm malzemeleri test etmek için tavsiye edilir. </li><li> Floresan kontrast madde green (ICG), steril koşullar altında hazırlanan indosiyanin. 0.5 mg / ml konsantrasyon kullanılır. Ikincisi agrega ICG neden olacak gibi steril distile su ve NaCl, kullanmak önemlidir. Hazırlanmasından sonra, çözüm floresan hızlı bozulmasını önlemek için bir karanlık, serin bir yerde saklanmalıdır. Not: ICG hazırladıktan sonra, eldiven değiştirmek için tavsiye edilir ICG, dökmek, hatta küçük bir miktar olarak görüntü kalitesini etkileyebilir. </li><li> Görüntüleme prosedürü karın açıldıktan sonra başlar. Ilgi alanı maruz kaldığında, kamera işletim alana manevra. Zoom ve odaklama steril koşullar altında ayarlanır. Ameliyathanede Işıklar floresan sinyal daha iyi algılanması için kapatılır. </li><li> 0.5 mg / ml ICG 1.0 mL 1,0 mL standart mavi boya (Patent mavi / bleu patente, Guerbet, Fransa) bir şırınga ile karıştırılır. Cerrah ajan dökülen engelleyen primer tümörün etrafında dört kadran kontrast madde enjekte eder. Not: cerrahi eldiven ICG dökülme durumunda, eldiven değiştirmek için gerekli. Hareketsiz görüntüler veya gerçek-zamanlı video lenf akışını ve floresan sentinel lenf nodu (Şekil 3) görünümünü elde edilir. Tüm resimler ve videolar direkt olarak bilgisayara kaydedilir. </li><li> Apart floresan SLN de ya bir gözle gama prob veya mavi renk, ya da her ikisi kullanılarak, standart protokole göre algılanır. Radyoaktif tracer genellikle sonra lymphoscintigram radyoaktif SLN tespiti için yapılır, ameliyattan bir gün önce uygulanır. Bu standart prosedür parçasıdır ve floresan görüntüleme protokolünde yeri yoktur. </li><li> SLN Gerçek-zamanlı eksizyonu, lenf düğümleri floresan ve mavi renk tarafından yönlendirilir. Kameranın gerçek zamanlı özelliği, herhangi bir kalıntı floresan veya floresan düğümleri beklenmedik lokalizasyonu için tespit etmek için yardımcı olur. </li><li> SLN eksizyonu sonra, ex vivo görüntüleri floresan sinyal varlığı için eksize tüm lenf düğümleri elde edilir. (Şekil 4), fotoğraf çekerken yüksek çözünürlük için maruz kalma süresi uzatılabilir. </li><li> Lenf nodlarının histopatolojik inceleme sentinel lenf nodu, tümör hücrelerinin varlığı ya da yokluğu ortaya koymaktadır. </li></ol> 4. Representative Sonuçlar Floresan lenf düğümleri yüksek bir sinyal-zemin oranı tespit edilebilir. Ayrıca, Lenf damarları yoluyla ICG akışı, gerçek zamanlı lenf nodu eşleşmesine olanak olarak izlenebilir. Sonuçlar muhtemelen algılama ulaşmak için farklı aydınlatma işlemleri gerektiren, düğümün derinliği artan etkilenebilir. Spesifik tümör hedefli floresan ajanlar Geleceği uygulama, bu teknoloji kullanarak kanser hücrelerinin ile pozitif SLN intraoperatif algılama sağlayabilir. <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="Şekil 1" /> Şekil 1. Sentinel lenf nodu (SLN) teorisi. SLN tümör ilk drenaj lenf nodu (ler). <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="Şekil 2" /> Şekil 2. Multispektral floresan kamera sistemi şematik resim (A). Ameliyathane kamera sistemi temel kurulum (B). <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="Şekil 3" /> Şekil 3. Vulva kanserinde lenf nodu Çokspektrumlu floresan görüntüleme in vivo bir lenf nodu (A) Renkli görüntü. Floresan in vivo aynı lenf nodu, (B) görüntüsü. Pseudocolor floresan görüntü, renkli görüntü (C) üzerine bindirilmiş. <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="Şekil 4" /> Şekil 4. Çokspektrumlu floresan görüntüleme serviks kanseri lenf nodu ex vivo bir lenf nodu ex vivo (A) Renkli görüntü. Aynı lenf nodu, ex vivo (B) Floresans görüntüsü. Pseudocolor floresan görüntü, renkli görüntü (C) üzerine bindirilmiş.

<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 ve GMvD prosedürü önce SurgOptix Inc bilimsel kurulu üyesi, araştırma protokolü, yerel Araştırma İnceleme Kurulu (KİK) ve hasta elde edilir bilgilendirilmiş onam tarafından onaylanmıştır.

Bu video jinekolojik onkoloji sentinel lenf nodu (SLN) intraoperatif tespiti için multispektral intraoperatif floresan görüntüleme teknolojisi uygulama göstermektedir. Metodoloji geleneksel SLN prosedürü üzerinde bazı avantajları vardır. Enjeksiyonu özellikle jinekolojik kanserlerde, cerrahi prosedürü yerine, daha fazla hasta dostu cerrahi öncesi bir gün, çok hasta anestezi ile kendini sırasında yer alıyor. Ayrıca, intraoperatif görüntüleme Geiger-vezne aracılığı ile dolaylı olarak lenf akışını ve drenaj desen SLN yerine, doğrudan görsel geribildirim ile cerrah sağlar. Serviks kanseri, lenf düğümleri pelvis derin nerede, bu algılama geliştirmek yardımcı olabilir. Bununla birlikte, en büyük önemi hedef görüntüleme doğru adım yaklaşımı yatıyor. Hedeflenen floresan kontrast ajanlar, özellikle tümör hücrelerinin yönelik tümör-spesifik antikorların veya substratları ile erimiş olabilir. Bu şekilde, intraoperatif floresan görüntüleme radikal onkolojik cerrahi mevcut uygulamayı değiştirmek için büyük bir potansiyele sahiptir.

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>
<tr>
<td>Multispectral fluorescence camera system </td>
<td>&nbsp;</td>
<td>Institute for Biological and Medical Imaging (IBMI), Technical University, Munich, Germany and SurgOptix Inc, USA</td>
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multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology

Hemen hemen bütün katı tümörlerde prognoz lenf nodu metastazı varlığı ya da yokluğuna bağlıdır. 1-3 Cerrahi tedavi en sık tümör tümör drenaj alanı tam bir lenfadenektomi ile birlikte radikal eksizyon birleştirir . Bununla birlikte, lenf nodlarının çıkarılması, enfeksiyon, yara ayrışması ve lenfödem nedeniyle artmış morbidite ile ilişkilidir. 4,5 alternatif olarak, sentinel lenf nodu (SLN) işlem tümörün ilk drenaj lenf nodu tespit etmek için onlarca yıl önce geliştirilmiştir lymphogenic yayılması durumunda 6, SLN etkilenen ilk lenf nodu (Şekil 1). Bu nedenle, SLN metastaz içermiyorsa, alt lenf düğümleri de tümörün metastaz ücretsiz ve kaldırılması gerekir. SLN prosedürü, meme kanseri ve melanom gibi birçok tümör tipleri için değil, aynı zamanda vulva ve serviks kanseri için tedavinin bir parçasıdır. SLN tespiti için 7 standart bir yöntem cerrahi öncesi radiocolloid bir gün peritümöral enjeksiyon yoluyla, intraoperatif ve renkli bir boya. Servikal ve vulvar kanser prosedürü dezavantajları birden fazla hasta için artan psikolojik sıkıntı önde gelen genital bölgede enjeksiyonlar, radyoaktif kolloid kullanımı. Multispektral floresan görüntüleme intraoperatif radiocolloid enjeksiyon için gerek kalmadan uygulanabilir gelişmekte olan bir görüntüleme yöntemidir. Intraoperatif floresan görüntüleme için, iki bileşenden ihtiyaç vardır: floresan intraoperatif görüntüleme için ajan ve nicel bir optik sistem. Fluorofor olarak kullanılan indosiyanin yeşili (ICG) var. ICG, on yıllar boyunca, kalp fonksiyonu, serebral perfüzyon ve karaciğer perfüzyon değerlendirmek için kullanılır olmuştur. 8 Bu farmako biyolojik güvenli bir profil ile inert bir ilaçtır. Yaklaşık 750 nm&#39;de heyecanlı zaman, bu, yakın-kızılötesi spektrumunda 800 nm civarında ışık yayar. Ismarlama multispektral floresan görüntüleme kamera sistemi kullanıldı. 9. Bu video makalenin amacı, servikal ve vulvar kanseri olan hastalarda intraoperatif floresan görüntüleme kullanarak SLN tespiti göstermektir. Floresan görüntüleme radiocolloid ve mavi boya oluşan standart prosedür ile birlikte kullanılır. Gelecekte, intraoperatif floresan görüntüleme geçerli yöntem yerine ve aynı zamanda, meme kanseri ve melanom gibi diğer göstergeler kolayca devredilemez.

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

Floresans görüntüleme, görüntü kılavuzluğunda cerrahi cerrahi onkoloji için umut verici bir yenilikçi bir yöntemidir. Bu video jinekolojik oncologicy görücüye olarak floresan görüntüleme ile sentinel lenf nodu tespiti için teknik prosedür açıklanmaktadır. Multispektral floresan kamera sistemi, floresan madde ile birlikte yeşil indosiyanin uygulanır.

Jinekolojik Onkoloji Sentinel lenf nodu intraoperatif Algılama için Multispektral Gerçek zamanlı Floresans Görüntüleme

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 Görüntüleme.  University Medical Center Groningen. Floresans görüntüleme, görüntü kılavuzluğunda cerrahi cerrahi onkoloji için umut verici bir yenilikçi bir yöntemidir. Bu video jinekolojik oncologicy görücüye olarak floresan görüntüleme ile sentinel lenf nodu tespiti için teknik prosedür açıklanmaktadır. Multispektral floresan kamera sistemi, floresan madde ile birlikte yeşil indosiyanin uygulanır.

Video: 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.

Intravital Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

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

Tıp

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.

Jinekolojik Kanserler için Stereotaktik Radyocerrahi

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.

Kantitatif Gerçek-zamanlı PCR (qPCR) tarafından Prostat tümörlerinde mikroRNA Algılama

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.

Hafif Endometriozis İntraoperatif Algılama: Periton Bütünlüğü Kaybı ile İlişkili Pelvik Ağrı Tespiti ve Tedavisi İçin Yeni Bir Paradigma

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.

Kanser Tedavisi İzleme Cerenkov Lüminesans Görüntüleme (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.

Boyuna, Non-invaziv için Eye Ön Kamara içine Nakli<em&gt; In vivo</emGerçek zamanlı Tek hücreli Çözünürlük&gt; Görüntüleme

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.

Kemoterapi kaynaklı Vasküler Toksisite - Gerçek zamanlı<em&gt; In vivo</em&gt; Gemi Düşüklüğü Görüntüleme

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.

İnsan periton boşluğuna bir Organotipik Kültür Yumurtalık Kanseri Yaygınlaştırılması Erken Adımlar Modelleme

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.

Tümör mikroçevresinde Gerçek zamanlı Multisellüler Dynamics Genişletilmiş Time-lapse intravital Görüntüleme

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.