Wir danken Frau Ina Wesselman für Training und Planung der OP-Personal.

1. Intraoperative Multispectral Fluoreszenz Kamera <ol><li> Eine maßgeschneiderte Kamera-System wurde entwickelt am Institut für Biologische und Medizinische Bildgebung (IBMI, Technische Universität / Helmholtz Zentrum München, Deutschland) in der Nähe in Zusammenarbeit mit SurgOptix (SurgOptix Inc, Redwood Shores, CA, USA) entwickelt. Der Aufbau der intraoperative multispektralen Fluoreszenz Kamera-System ist in Abbildung 2 dargestellt. </li><li> Licht gelangt durch ein System von Optik und in sichtbares Licht, Licht bei der Emission (Fluoreszenz) Wellenlängenbereich und Licht bei der Anregungswellenlänge Band getrennt. Diese Bündel werden von CCD-Kameras (CCD) (Abbildung 2) erkannt. Multi-Spektral-Signale von allen Kameras verarbeitet werden, um für die Artefakte zu korrigieren und Ertrag stimmt quantitative Fluorochrom bio-Verteilung. Farbe und Fluoreszenz-Signale können als separate Bilder auf externen Monitoren angezeigt werden oder überlagert in einem Bild. </li><li> Die Anregungs-und Emissions-Filter der Kamera werden in die Messung hier eingestellt bei 750 nm und 800 ± 20 nm bzw. zu sammeln. </li></ol> 2. Optischen Kontrastmittels Vorbereitung <ol><li> Die fluoreszierenden Kontrastmittels Indocyaningrün (ICG, Pulsion AG, München, Deutschland) unter sterilen Bedingungen hergestellt wird. Eine Konzentration von 0,5 mg / mL eingesetzt. Es ist wichtig, steriles destilliertes Wasser und nicht Natriumchlorid (NaCl) zu verwenden, da diese die ICG zu aggregieren führen wird. </li><li> Nach der Zubereitung muss die Lösung in einem dunklen, kühlen Ort, um schnelle Verschlechterung der Fluoreszenzintensität durch Bleichen zu vermeiden gespeichert werden. Hinweis: Von diesem Punkt an halten die ICG vor Licht geschützt. </li></ol> 3. Intraoperative Imaging - Setup und Injektion von ICG <ol><li> Die Kamera ist so positioniert und initiiert im Operationssaal vor der Operation, um Interferenzen mit der Standard-chirurgischen Eingriff zu minimieren, und mit dem High-Definition-Bildschirme (Abbildung 2B). </li><li> Die Kamera ist in Standard-sterilen Tüchern (Carl Zeiss Vision BV, Sliedrecht, den Niederlanden, OPMI Drape REF 306071) durch den Prüfarzt in sterile OP Kleidung bedeckt sind. </li><li> Hinweis: wird darauf geachtet, nur klare Desinfektionsmittel zu verwenden, als farbige Desinfektionsmittel oft autofluoreszierenden und könnte mit dem bildgebenden Verfahren einmischen. Darüber hinaus können sterilen Tüchern und chirurgischen Marker autofluoreszierenden. Es wird empfohlen, alle Materialien im OP und das OP-Feld für die Autofluoreszenz vor der Operation zu testen. </li><li> Die fluoreszierenden Kontrastmittels Indocyaningrün (ICG) unter sterilen Bedingungen hergestellt wird. Eine Konzentration von 0,5 mg / mL eingesetzt. Es ist wichtig, steriles destilliertes Wasser und nicht NaCl, als dieser die ICG zu aggregieren führen wird. Nach der Zubereitung muss die Lösung in einem dunklen, kühlen Ort, um schnelle Verschlechterung der Fluoreszenz-Intensität zu vermeiden gespeichert werden. Hinweis: Es ist ratsam, Handschuhe zu ändern, nachdem der Vorbereitung ICG, wie auch eine kleine Menge von Spill von ICG kann die Bildqualität beeinflussen. </li><li> Die bildgebenden Verfahren beginnt nach Eröffnung der Bauchhöhle. Wenn der Bereich von Interesse ausgesetzt ist, ist die Kamera in das Operationsfeld manövriert. Der Zoom und Fokus werden unter sterilen Bedingungen angepasst. Leuchtet im Operationssaal sind off für eine bessere Erkennung der Fluoreszenz-Signal geschaltet. </li><li> 1,0 ml von 0,5 mg / mL ICG wird mit 1,0 ml der Standard-blauen Farbstoff (Patent blau / bleu patente, Guerbet, Frankreich) in einer Spritze gemischt. Der Chirurg spritzt das Kontrastmittel in vier Quadranten um den Primärtumor und verhindert Verschütten des Agenten. Hinweis: Im Falle eines Übergreifens der ICG auf dem OP-Handschuhe, ist es notwendig, Handschuhe zu ändern. Standbilder oder Echtzeit-Videos sind von den Lymphfluss und das Aussehen des fluoreszierenden Sentinel-Lymphknoten (Abbildung 3) erworben. Alle Bilder und Videos werden direkt auf dem Computer gespeichert. </li><li> Abgesehen von Fluoreszenz, ist das SLN auch nach dem Standard-Protokoll erkannt wird, entweder mit Hilfe eines Gamma-Sonde oder durch visuelle Inspektion für eine Blaufärbung, oder beides. Die radioaktiven Tracer ist in der Regel verwaltet einen Tag vor der Operation, nach der ein lymphoscintigram für den Nachweis eines radioaktiven SLN durchgeführt wird. Dies ist Teil des Standard-Verfahren und hat keinen Platz in der Fluoreszenz-Imaging-Protokoll. </li><li> Real-time Exzision des SLN ist sowohl von Fluoreszenz-und Blaufärbung der Lymphknoten geführt. Die Echtzeit-Funktion der Kamera hilft, für jeden Rest Fluoreszenz oder unerwartete Lokalisierungen von fluoreszierenden Knoten zu erkennen. </li><li> Nach Exzision des SLN sind ex vivo Bilder aller herausgeschnitten Lymphknoten auf das Vorhandensein eines Fluoreszenzsignals erworben. Die Belichtungszeit kann für eine höhere Auflösung verlängert werden bei der Aufnahme von Standbildern (Abbildung 4). </li><li> Histopathologische Untersuchung der Lymphknoten zeigt das Vorhandensein oder Fehlen von Tumorzellen in der Sentinel-Lymphknoten. </li></ol> 4. Representative Ergebnisse Fluorescent Lymphknoten können mit einem hohen Signal-zu-Untergrund-Verhältnis nachgewiesen werden. Außerdem kann Strom von ICG durch Lymphgefäße überwacht ermöglicht Echtzeit-Lymphknoten-Mapping werden. Die Ergebnisse können durch eine Erhöhung Tiefe des Knotens beeinflusst werden, möglicherweise die unterschiedliche Ausleuchtung Verfahren zur Erkennung zu erreichen. Zukünftige Anwendung der spezifischen Tumor gezielt fluoreszierende Stoffe können vorsehen, intraoperative Nachweis eines positiven SLN mit Krebszellen mit dieser Technologie. <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Sentinel-Lymphknoten (SLN) Theorie. Die SLN ist der erste Lymphknoten (s) aus dem Tumor. <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schematische Darstellung der multispektralen Fluoreszenz-Kamera-System (A). Der grundsätzliche Aufbau des Kamerasystems in den Operationssaal (B). <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="Abbildung 3" /> Abbildung 3. Multispectral Fluoreszenz-Imaging von einem Lymphknoten in der Vulva Krebs. Farbbild eines Lymphknotens in vivo (A). Fluoreszenz-Bild der gleichen Lymphknoten, in vivo (B). Pseudofarben Fluoreszenzbild auf dem Farbbild (C) überlagert. <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="Abbildung 4" /> Abbildung 4. Multispectral Fluoreszenz-Imaging von einem Lymphknoten ex vivo bei Gebärmutterhalskrebs. Farbbild von einem Lymphknoten ex vivo (A). Fluoreszenz-Bild der gleichen Lymphknoten, ex vivo (B). Pseudofarben Fluoreszenzbild auf dem Farbbild (C) überlagert.

<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 und GMVD sind Mitglied des wissenschaftlichen Beirats der SurgOptix Inc. Vor dem Eingriff wird die Forschungs-Protokoll, das von der lokalen Investigational Review Board (IRB) und informierte Einwilligung des Patienten erfasst wird genehmigt.

Dieses Video zeigt die Anwendung des multispektralen intraoperative Fluoreszenz-Imaging-Technologie zur intraoperativen Detektion des Sentinel-Lymphknoten (SLN) in der gynäkologischen Onkologie. Die Methodik hat gewisse Vorteile gegenüber den herkömmlichen Verfahren SLN. Die Einspritzung erfolgt während des chirurgischen Eingriffs selbst mit dem Patienten narkotisiert eher als einen Tag vor der Operation, die mehr Patienten-freundlich, vor allem in gynäkologischen Krebserkrankungen. Darüber hinaus bietet die intraoperative Bildgebung der Chirurg mit direktem visuellen Feedback von den Lymphfluss und ihr Einzugsgebiet Muster auf die SLN, anstatt indirekt über einen Geiger-teller. In Gebärmutterhalskrebs, wo Lymphknoten tief im Becken befindet, kann dies dazu beitragen, Detektion. Dennoch liegt die größte Bedeutung in der Step-up-Ansatz zur gezielten Bildgebung. Gezielte fluoreszierende Kontrastmittel können mit Tumor-spezifischen Antikörpern oder Substrate spezifisch auf Tumorzellen gerichtet abgesichert werden. Auf diese Weise hat intraoperative Fluoreszenz-Imaging ein großes Potenzial, radikal verändern die derzeitige Praxis der onkologischen Chirurgie.

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>
<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

Die Prognose in nahezu allen soliden Tumoren hängt von der Anwesenheit oder Abwesenheit von Lymphknotenmetastasen. 1-3 chirurgische Behandlung meist kombiniert radikale Entfernung des Tumors mit einem vollen Lymphadenektomie im Einzugsgebiet des Tumors. Allerdings Entfernung der Lymphknoten mit erhöhter Morbidität aufgrund einer Infektion, Zersetzung der Wunde und Lymphödem verbunden ist. 4,5 Als Alternative wurde die Sentinel-Lymphknoten-Verfahren (SLN) vor einigen Jahrzehnten zu den ersten Lymphknoten aus dem Tumor zu erkennen entwickelt . 6 Im Falle einer lymphogenen Verbreitung, ist der SLN der erste Lymphknoten, der betroffen ist (Abbildung 1). Wenn also die SLN enthält keine Metastasen, wird nachgeschalteten Lymphknoten auch von Tumormetastasen frei und müssen nicht entfernt werden. Die SLN Verfahren ist Teil der Behandlung für viele Tumorarten wie Brustkrebs und Melanomen, sondern auch für Krebs der Vulva und Zervix. 7 Der aktuelle Standard-Methodik für die SLN-Detektion wird durch peritumoral Injektion von Radiokolloids 1 Tag vor der Operation, und einen Farbstoff intraoperativ. Nachteile des Verfahrens in Hals-und Vulvakarzinome sind mehrere Injektionen in den Genitalbereich, was zu einer erhöhten psychischen Belastung für den Patienten und die Verwendung von radioaktivem Kolloid. Multispectral Fluoreszenz-Bildgebung ist eine neue Bildgebungsverfahren, die intraoperativ kann, ohne die Notwendigkeit für die Injektion von Radiokolloids angewendet werden. Für intraoperative Fluoreszenz-Bildgebung werden zwei Komponenten benötigt: ein fluoreszierendes Mittel und eine quantitative optische System zur intraoperativen Bildgebung. Als Fluorophor wir verwendet haben Indocyaningrün (ICG). ICG ist seit vielen Jahrzehnten eingesetzt, um die Herzfunktion, zerebrale Perfusion und Leberperfusion zu beurteilen. 8 Es ist ein inertes Medikament mit einem sicheren Pharmako-biologische Profil. Wenn bei rund 750 nm angeregt, sendet es in der Nah-Infrarot-Spektrums um 800 nm-Licht. Eine maßgeschneiderte multispektralen Fluoreszenz-Imaging-Kamera-System verwendet wurde. 9. Das Ziel dieses Video-Artikels ist es, den Nachweis der SLN mit intraoperative Fluoreszenz-Bildgebung bei Patienten mit Hals-und Vulvakarzinome demonstrieren. Fluoreszenz-Bildgebung ist in Verbindung mit dem Standard-Verfahren verwendet, bestehend aus Radiokolloids und einen blauen Farbstoff. In Zukunft könnte intraoperative Fluoreszenz-Imaging ersetzen die aktuelle Methode und ist auch leicht übertragbar auf andere Indikationen wie Brustkrebs und Melanomen.

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

Fluoreszenz-Bildgebung ist eine viel versprechende innovative Modalität für die Bild-Chirurgie in der chirurgischen Onkologie. In diesem Video, das wir beschreiben die technischen Verfahren zur Detektion der Sentinel-Lymphknoten mittels Fluoreszenz-Bildgebung als in gynäkologischen oncologicy präsentiert. Eine multispektrale Fluoreszenz-Kamera-System, zusammen mit dem fluoreszierenden Indocyaningrün, angewendet wird.

Multispectral Real-time Fluoreszenz-Imaging für die intraoperative Detektion des Sentinel Lymph Node in Gynäkologische Onkologie

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 Aufrufe.  University Medical Center Groningen. Fluoreszenz-Bildgebung ist eine viel versprechende innovative Modalität für die Bild-Chirurgie in der chirurgischen Onkologie. In diesem Video, das wir beschreiben die technischen Verfahren zur Detektion der Sentinel-Lymphknoten mittels Fluoreszenz-Bildgebung als in gynäkologischen oncologicy präsentiert. Eine multispektrale Fluoreszenz-Kamera-System, zusammen mit dem fluoreszierenden Indocyaningrün, angewendet wird.

Serie: 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.

Evaluation von Nanopartikel-Aufnahme in Tumoren in Echtzeit über 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 ...

Forschung

Medizin

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.

Stereotaktische Radiochirurgie für Gynäkologische Krebs

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.

MicroRNA-Detektion in Prostatatumoren durch 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 ...

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.

Intraoperative Detektion von Subtle Endometriosis: A Novel Paradigm for Detection und Behandlung von Pelvic Pain mit dem Loss of Peritoneal Integrity-Zustellbare

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.

Cerenkov Lumineszenz Imaging (CLI) for Cancer Therapy Überwachung

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.

Transplantation in die Vorderkammer des Auges für Längs-, Non-invasive<em&gt; In vivo</em&gt; Imaging mit Single-cell Auflösung in Echtzeit

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.

Chemotherapie-induzierte vaskuläre Toxizität - Echtzeit-<em&gt; In-vivo-</em&gt; Imaging von 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 ...

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.

Modellierung der frühen Schritte der Eierstockkrebs Verbreitung in einer organotypischen Kultur der Menschen Peritonealhöhle

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.

Erweiterte Zeitraffer-Intravital Imaging von Echtzeit-Vielzellige Dynamics im Tumormikromilieu

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.