우리는 또는 직원의 훈련과 계획에 대해 부인이나 Wesselman에게 빚을 졌네.

1. Intraoperative Multispectral 형광 카메라 <ol><li> 맞춤 만든 카메라 시스템은 SurgOptix (SurgOptix 병원, 레드우드 쇼어스, CA, 미국)와 공동으로 가까이에서 생물학 및 의료 이미징에 대한 연구소 (IBMI, 기술 대학 / Helmholtz 젠트룸, 뮌헨, 독일)에서 개발에서 개발되었습니다. intraoperative multispectral 형광 카메라 시스템의 설정은 그림 2에 표시됩니다. </li><li> 빛이 광학의 시스템을 통해 전달하고 가시 광선의 방출 (형광) 파장 대역에서 가볍고 여기 파장 대역의 빛을으로 구분합니다. 이러한 번들은 CCD - 카메라 (CCD) (그림 2)에 의해 감지됩니다. 모든 카메라에서 다중 스펙트럼 신호는 유물에 대한 정확하고 진정한 양적 fluorochrome의 생체 분포를 얻을 수 있도록 처리됩니다. 색상과 형광 신호를 외부 모니터에 별도의 이미지로 표시되거나 하나의 이미지에 겹쳐 수 있습니다. </li><li> 카메라의 여기 및 발광 필터는 각각 750 nm의 800 ± 20 nm의에서 수집하는 측정 여기에서 설정합니다. </li></ol> 2. 광학 대비 에이전트 준비 <ol><li> 형광 대비 에이전트가 녹색이 (ICG, Pulsion AG, 뮌헨, 독일) 무균 조건 하에서 준비 indocyanine. 0.5 MG / ML의 농도가 사용됩니다. 후자가 집계하는 ICG의 원인이되므로, 살균 증류수 아니라 나트륨 염화물 (NaCl)를 사용하는 것이 중요합니다. </li><li> 준비 후, 솔루션은 표백하여 형광 강도의 빠르게 악화를 피하기 위해 어둡고 시원한 장소에 보관해야합니다. 참고 :이 시점에서 앞으로 빛으로부터 보호 ICG을 유지. </li></ol> 3. Intraoperative 이미징 - 설치 및 ICG의 분사 <ol><li> 카메라 위치 및 표준 수술로 간섭을 최소화하기 위해 이전에 수술로 운영 극장에서 시작하고 고해상도 화면 (그림 2B)로 연결되어 있습니다. </li><li> 카메라는 멸균 또는 의류에 수사관에 의해 표준 살균 커튼 (칼 자이스 혈구 비전 BV, Sliedrecht, 네덜란드, OPMI의 드레이프의 REF 306071)에 덮여있다. </li><li> 참고 : 색상 소독 요원은 종종 autofluorescent되고 이미징 절차 방해 하듯이, 오직 맑은 소독 요원을 사용하는 신경 이루어집니다. 또한, 무균 커튼, 외과 마커 autofluorescent 수 있습니다. 그것은 이전 수술 autofluorescence에 대한 OR 및 운영 분야에서 사용되는 모든 자료를 테스트하는 것이 좋습니다. </li><li> 형광 대비 에이전트 녹색 (ICG)을 무균 조건 하에서 준비 indocyanine. 0.5 MG / ML의 농도가 사용됩니다. 후자가 집계하는 ICG의 원인이되므로 살균 증류수가 아닌 NaCl을 사용하는 것이 중요합니다. 준비 후 솔루션은 형광 강도의 빠르게 악화를 피하기 위해 어둡고 시원한 장소에 보관해야합니다. 참고 :이 ICG를 준비 후 장갑을 변경하는 것이 좋습니다 ICG의 유출의도 소량으로 이미지 품질에 영향을 미칠 수 있습니다. </li><li> 이미지 절차는 복부를 열고 후에 시작됩니다. 관심 분야가 노출되면 카메라가 작동 필드에 였는데입니다. 줌 및 초점은 무균 조건 하에서 조정됩니다. 운영 극장에서 조명은 형광 신호를 더 잘 감지 꺼져 있습니다. </li><li> 0.5 MG / ML ICG 1.0 ML 한 주사기에서 표준 청색 색소 (특허 블루 / 블루 patenté, Guerbet, 프랑스)의 1.0 ML로 혼합합니다. 의사는 에이전트 유출을 방지, 기본 종양 주위에 네 quadrants에 대비 에이전트를 삽입합니다. 참고 : 수술 장갑에 ICG의 유출의 경우, 그것이 장갑을 변경할 필요가 있습니다. 스틸 이미지 또는 실시간 동영상은 림프의 흐름과 형광 센티널 림프 노드 (그림 3)의 모양을 찾았습니다. 모든 이미지 및 동영상은 직접 컴퓨터에 저장됩니다. </li><li> 외에도 형광에서 SLN는 역시 감마 프로브 또는 육안 검사에 의해 파란색으로 변색에 대한, 또는 둘 모두를 사용하여 표준 프로토콜에 따라 검색합니다. 방사성 추적자는 일반적으로 lymphoscintigram가 방사성 SLN의 탐지를 위해 수행하는 후 수술을하기 전에 일일 관리합니다. 이것은 표준 절차의 한 부분이며, 형광 이미징 프로토콜에 자리가 없습니다. </li><li> SLN의 실시간 절단은 림프절의 형광 및 파랑 변색 모두로 안내합니다. 카메라의 실시간 기능은 잔여 형광이나 형광등 노드의 예기치 localizations에 대한 감지하는 데 도움이됩니다. </li><li> SLN의 절단 후, 전 생체내 이미지는 형광 신호의 존재에 대한 모든 excised 림프절의 인수입니다. (그림 4) 정지 영상 캡처하면 노출 시간은 더 높은 해상도를 위해 연장하실 수 있습니다. </li><li> 림프절의 Histopathological 심사는 센티널 림프절에 종양 세포의 존재 또는 부재를 보여준다. </li></ol> 4. Representative 결과 형광등 림프절은 높은 신호 대 배경 비율로 검색하실 수 있습니다. 또한, 림프 혈관을 통해 ICG의 흐름이 실시간으로 림프절 매핑을 허용 모니터링할 수 있습니다. 결과는 가능성이 다른 조명 절차는 검출을 달성하기 위해 필요한, ​​노드의 증가 깊이 영향을받을 수 있습니다. 특정 종양 대상으로 형광 대리인의 미래 응용 프로그램은이 기술을 사용하여 암세포와 함께 긍정적인 SLN의 intraoperative 감지를 제공할 수 있습니다. <img src="/files/ftp_upload/2225/2225fig1.jpg" alt="그림 1" /> 그림 1. 센티널 림프절 (SLN) 이론. SLN은 종양에서 첫 번째 배수 림프절 (들)입니다. <img src="/files/ftp_upload/2225/2225fig2.jpg" alt="그림 2" /> 그림 2. multispectral 형광 카메라 시스템의 개략도 그림 (A). 운영 극장에서 카메라 시스템의 기본 설정 (B). <img src="/files/ftp_upload/2225/2225fig4.jpg" alt="그림 3" /> 그림 3. vulvar 암에서 림프절의 Multispectral 형광 이미징. 생체내 (A)에서 림프절의 컬러 이미지. 생체내에서 동일한 림프 노드 (B)의 형광 이미지. Pseudocolor 형광 이미지가 컬러 이미지 (C)에 겹쳐. <img src="/files/ftp_upload/2225/2225fig5.jpg" alt="그림 4" /> 그림 4. 자궁 경부암에서 림프절의 전의 생체내의 Multispectral 형광 이미지. 림프 노드 예 생체내 (A)의 컬러 이미지. 같은 림프절, 전직 생체내 (B)의 형광 이미지. Pseudocolor 형광 이미지가 컬러 이미지 (C)에 겹쳐.

<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 및 GMvD는 전에 절차 SurgOptix 주식 회사의 과학위원회의 회원, 연구 프로토콜은 로컬 Investigational 검토위원회 (IRB)와 환자가 취득의 정보를 동의에 의해 승인됩니다.

이 동영상은 gynecologic 종양학의 센티널 림프절 (SLN)의 intraoperative 감지 multispectral intraoperative 형광 이미징 기술의 응용 프로그램을 보여줍니다. 방법론은 기존의 SLN 절차를 통해 특정 장점이 있습니다. 주사 특히 gynecologic의 암에서 수술보다는 더 많은 환자 친화 전에 수술로 하루 이상 anesthetized 환자와 자체 동안에 이루어진다. 또한, intraoperative 이미지는 가이거 - 텔러를 통해 간접적으로 림프의 흐름과 배수 패턴 SLN하기보다는 직접 시각적 피드백과 의사를 제공합니다. 자궁 경부암에서 림프절이 골반 깊은 위치에, 이것은 검색을 개선하는 데 도움이 될 수 있습니다. 그럼에도 불구하고, 가장 중요한 타겟이 영상 향한 단계까지 접근에있다. 타겟 형광 대비 요원은 특히 종양 세포에서 감독 종양 특정 항체 또는 기판과 융합 수 있습니다. 이러한 방법으로, intraoperative 형광 이미징은 크게 oncologic 수술의 현재 관행을 변경하기 위해 큰 잠재력이있다.

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

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
</tr>
<tr>
<td>Patent blue / Bleu patent&eacute;</td>
<td>&nbsp;</td>
<td>Guerbet, Paris, France</td>
<td>12322784</td>
<td>&nbsp;</td>
<tr>
<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>
<td>prototype</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology

거의 모든 고체 종양의 예후는 림프 노드 metastases의 존재 또는 부재에 따라 달라집니다. 1-3 외과 치료가 가장 자주 종양의 배수 지역에 전체 lymphadenectomy와 종양의 급진적인 절단을 조합한 것입니다. 그러나, 림프절의 제거가 감염, 상처 분해 및 lymphedema으로 인해 증가 병적 상태와 연결되어 있습니다. 대안으로 4,5, 센티널 림프절 절차 (SLN)은 종양에서 첫 번째 배수 림프절을 감지하는 방법에는 여러 수십 년 전에 개발되었습니다 lymphogenic 보급의 경우. 6 SLN는 영향을받는 첫 번째 림프절 (그림 1)입니다. 따라서 SLN가 metastases를 포함하지 않는 경우, 하류 림프절은 종양 metastases에서 무료로하고 제거되지 않도록해야합니다. SLN 절차는 많은 종양 유형, 유방암과 흑색증 같은뿐만 아니라 외음부와 자궁 경부의 암 치료의 일부입니다. SLN 예방을위한 7 현재 표준 방법론 전에 수술 radiocolloid 하루 peritumoral 주입하는 것입니다, intraoperatively와 컬러 염료. 자궁 경부 및 vulvar 암의 절차의 단점은 여러 환자 증가 심리적 고통에 이르는 성기 영역에서 주사, 그리고 방사성 콜로이드를 사용하고 있습니다. Multispectral 형광 이미징은 radiocolloid의 주입을위한 필요없이 intraoperatively 적용할 수있는 새로운 이미징 양상이다. intraoperative 형광 이미징을위한 두 개의 구성 요소가 필요합니다 : 형광등 에이전트 및 intraoperative 이미징을위한 광학 시스템을 양적. 형광단로서 우리는 녹색 (ICG)를 indocyanine 사용했습니다. ICG는 심장 기능, 뇌성 재관류 및 간 재관류를 평가하기 위해 여러 수십 년 동안 사용되었습니다. 8 그것은 안전 pharmaco - 생물 학적 프로파일 불활성 약물이다. 750 nm의 주위에 흥분 때, 800 nm의 주위에 가까운 적외선 스펙트럼의 빛을 방출. 맞춤 제작 multispectral 형광 이미징 카메라 시스템이 사용되었다. 9. 이 비디오 문서의 목적은 자궁과 vulvar 암 환자의 intraoperative 형광 이미징을 사용하여 SLN의 검출을 설명하는 것입니다. 형광 이미징은 radiocolloid과 청색 색소의 구성, 표준 절차와 함께 사용됩니다. 앞으로 intraoperative 형광 이미징은 현재의 방법을 대체할 수 있으며 유방암과 흑색증 같은 다른 징후 쉽게 양도할 수 있습니다.

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

형광 이미징은 외과 종양학의 이미지 유도 수술 유망 혁신적인 양상이다. 이 비디오에서 우리는 gynecologic oncologicy에 전시로 형광 이미징을 사용하여 센티널 림프절의 탐지에 대한 기술적인 절차를 설명합니다. multispectral 형광 카메라 시스템이 함께 형광 에이전트 녹색 indocyanine, 적용됩니다.

Gynecologic 종양학의 센티널 림프절의 Intraoperative 감지 Multispectral 실시간 형광 이미징

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

23.1K 조회수.  University Medical Center Groningen. 형광 이미징은 외과 종양학의 이미지 유도 수술 유망 혁신적인 양상이다. 이 비디오에서 우리는 gynecologic oncologicy에 전시로 형광 이미징을 사용하여 센티널 림프절의 탐지에 대한 기술적인 절차를 설명합니다. multispectral 형광 카메라 시스템이 함께 형광 에이전트 녹색 indocyanine, 적용됩니다.

비디오: 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 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

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

연구

의학

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.

Gynecologic 암에 대한 Stereotactic Radiosurgery의

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.

정량 실시간 PCR (qPCR)에 의한 전립선 종양에 MicroRNA 감지

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.

미묘한 Endometriosis의 Intraoperative 감지 : 복막 무결성의 손실과 관련된 골반 통증의 감지 및 치료를위한 소설 패러다임

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.

암 치료 모니터링을위한 Cerenkov 발광 이미징 (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.

길이, 비 침습적을위한 안구의 앞쪽 상공 회의소에 이식<em&gt; 생체에</em실시간으로 단일 셀 해상도&gt; 영상

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.

화학 요법에 의한 혈관 독성 - 실시간<em&gt; 생체 내</em&gt; 선박 손상의 영상

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.

인간의 복강의의 Organotypic 문화 난소 암 보급의 초기 단계 모델링

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.

종양 미세 환경에서 실시간 다세포 역학의 확장 시간 경과 생체 내에 영상

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.