这项研究是由美国国防部的乳腺癌研究计划下授予号（ASH，W81XWH-13-1-0010），NIH CA100324，PPG CA100324，和综合成像计划的支持。

中描述的所有程序必须按照与使用脊椎动物，包括医药机构动物护理和使用委员会的爱因斯坦医学院的事先批准的准则和规定进行。 1.生成荧光标记的肿瘤及肿瘤相关巨噬细胞<ol><li>通过杂交自发，原地，遗传工程小鼠乳腺癌模型中，小鼠乳腺肿瘤病毒长末端重复与荧光报告[ 即转基因小鼠驱动多瘤中间T抗原（MMTV-PyMT），增强型绿色荧光产生的荧光标记的肿瘤细胞蛋白（EGFP），增强型青色荧光蛋白（ECFP）或者Dendra2] -8,9-。 </li><li>在PyMT动物荧光标记的巨噬细胞用荧光通过穿越myeloid-和巨噬细胞特异性荧光记者遗传工程小鼠模型[即 MacGr标记的肿瘤细胞EEN 15或MacBlue小鼠CSF1R -GAL4VP16 / UAS-ECFP 16]。 </li><li>可替代地，通过荧光标记PyMT肿瘤细胞（同基因），人乳腺癌细胞系或原代人类患者来源的肿瘤（异种移植物）11,17的原位移植产生荧光标记的肿瘤。 <ol><li>植入荧光标记PyMT肿瘤细胞与荧光蛋白标记的骨髓细胞同系小鼠以生成具有荧光标记的肿瘤细胞和骨髓细胞13的动物。 </li></ol></li><li>注入100微升的10毫克/公斤的荧光70 kDa的葡聚糖通过静脉内（IV）尾静脉进入重症联合免疫缺陷（SCID）小鼠与人细胞系或来源于患者的原发肿瘤荧光标记的巨噬细胞2小时之前开始的异种移植肿瘤的活体成像。 </li></ol> 2.显微镜设置和成像准备注:此过程描述的设立对多光子显微镜8活体成像。 <ol><li>打开所有显微镜和激光组件，包括双光子激光器和检测器。 </li><li>打开加热框到30℃预温阶段。这个步骤是用于维持动物的生理温度是至关重要的。 </li><li>有关倒置显微镜的地方在显微镜舞台上的定制阶段插入使用。自定义插入是"加工成适合在舞台插入空间和一个1厚铝"直径的通孔的中心成像的1/8片。 <ol><li>擦拭用70％乙醇和空气干燥的显微镜载物台和阶段插入。 </li><li>放置在20X，1.05 NA显微镜物镜一大滴的水，以保持与玻璃盖光学接触。 </li><li>地方盖玻璃（＃1.5厚度）在显微镜舞台上插入成像端口。安全与实验室胶带的地方。 </li><li>用打孔器打孔2厘米×2厘米的洞以灵活的橡胶垫，并将与在自定义阶段插入孔对准垫孔显微镜阶段的橡胶垫。 </li></ol></li></ol> 3.尾静脉导管的制备及试剂成像注射期间<ol><li>切聚乙烯（PE）管30厘米长。 </li><li>使用镊子，移动31克针金属针来回，直到它打破关闭塑料接头的。 </li><li>使用镊子，插入分离针的钝端进入PE管材。 </li><li>插入一个31克针进入PE管的另一端。 </li><li>填用无菌磷酸盐缓冲盐水（PBS）1毫升注射器，并将其插入到31克针和刷新所有空气从管和针用PBS中。 PBS用于维护水合和血液的渗透压9,18，也可以使用然而等渗盐水。 </li><li>填用200μl的3毫克1毫升注射器/ kg的155 kDa的葡聚糖 - 四甲基罗丹明（TMR）或血管标记的量子点。 </li><li>制备任何其它可注射的蛋白质，例如在1毫升注射器置于冰上血管内皮生长因子A（VEGFA 165）的一个165氨基酸同种型。注入0.2毫克/千克VEGFA 165 19以0.05毫克/毫升的浓度。 </li></ol> 4.插入尾留置静脉置管<ol><li>使用感应腔室与O 2作为载气的5％异氟烷放置麻醉下的动物。 </li><li>鼠标转移到外科平台一旦完全麻醉，并且不向脚趾捏响应。 </li><li>开异氟烷麻醉线到外科平台，关闭麻醉线到感应腔室，并放置在动物的鼻子的鼻锥体。从5％减小异氟醚为2.5％。 </li><li>应用眼药膏鼠标，以防止在麻醉过程中干涩的眼睛。 </li><li>热的加热灯下动物另外的2分钟，以增加流通尾巴作为循环深麻醉放缓。 </li><li>消毒用70％乙醇的小鼠尾静脉。 </li><li>插入从第3节中构建到动物的侧面尾静脉导管的31克针尖和推针在2-3毫米。 </li><li>向后拉动注射器见血，确保导管放置在尾静脉中。 </li><li>使用放置在针实验室带的1cm长，以代替平行针保持沿尾部的长度的静脉。 </li></ol> 5.皮瓣手术过程揭露乳腺肿瘤<ol><li>与手术平台上的动物拭肿瘤并用70％乙醇腹面。注意:如所描述的，不进行该过程完全在无菌条件下。对于长成像会议，其中感染可成为一个混杂因素，建议使用正确的无菌技术。 </li><li>用免缝勒镊子，提起腹侧皮肤，并用无菌剪刀使一个皮下切口沿腹中线大约为1厘米的长度。避免切口时刺破腹膜。 </li><li>轻轻切结缔组织附着的乳腺肿瘤从腹膜掉，露出乳房肿瘤。 </li><li>用剪刀和镊子，轻轻取出，并从肿瘤的暴露表面切去筋膜和脂肪，同时保持肿瘤脉管系统的完整性和减少出血。这一步骤是在维持肿瘤的肿瘤架构和脉管供应的关键。 </li></ol> 6.动物准备显微镜<ol><li>动物转移到预热的成像级与暴露的肿瘤放置在盖玻片上的阶段插入在用于成像的显微镜物镜。照顾到与动物轻轻转移尾静脉导管，以便不去除针头。 </li><li>第广场Ë麻醉鼻锥在动物的鼻子，以保持麻醉盘以3％异氟醚。 </li><li>使肿​​瘤掌握在橡胶垫的孔，并与该玻璃盖玻片接触动物的位置。 </li><li>用两个橡胶垫到位轻轻握住肿瘤，并将其固定在显微镜阶段，实验室的磁带，以减少图像采集过程中的运动。 </li><li>填补用PBS橡胶垫的腔室，以保持组织水合和保持与盖玻片光学接触。 </li><li>开始脉搏血氧仪探头监测动物的生命体征。附加的夹子传感器动物的大腿。备选地，可以使用被配置为套在脖子上的套环。 </li><li>放置加热箱在动物保持30℃20。 </li><li>异氟醚的水平慢慢降低到0.5-1％维持麻醉，维持血流。 </li></ol> 7.图像采集和Fluoresc注射耳鼻喉科染料和注射蛋白质<ol><li>使用显微镜目镜聚焦于肿瘤的表面上的荧光肿瘤细胞。 </li><li>通过用流动的血管发现区域选择感兴趣的区域。感兴趣的区域的与流血管的选择是用于评估肿瘤血管的关键。 </li><li>一旦感兴趣区域已被选择切换显微镜到多光子成像模式。 </li><li>设置Z系列的上限和下限。通过使用焦点调整到目标移动到所需的开始位置和点击的Z位置"顶部"按钮组的z系列的上限。通过在二次谐波产生（SHG）信道的肿瘤表面可视化胶原纤维网络并当无细胞，但只胶原纤维是可见的观测确定的z系列的上限。 <ol><li>由物镜移动到所希望的成像深度设置Z系列的下限（typicaLLY 50 - 150微米），并单击Z位置"底部"按钮。 </li><li>通过键入所需的值到步长字段设置为步长。确定基于考虑分辨率和采集时间步长（通常为2微米，用于高分辨率三维重建和5微米其他）。 </li></ol></li><li>通过切换到时间推移面板和输入所需的经过时间进入时间推移字段中设置的时间推移的时间间隔。为获得最佳的时间分辨率的时间间隔设置为0秒，连续成像。注意:对于长期延时成像建议的时间间隔设置为10秒采集之间以补充目标浸没液体。 </li><li>一旦成像参数已经确定，缓缓注入155 kDa的葡聚糖TMR。 <ol><li>在尾静脉导管除去，用PBS注射器，用含155 kDa的葡聚糖的TMR注意不要引入任何气泡进入里注射器替换东北。 </li><li>缓缓注入155 kDa的葡聚糖的TMR到小鼠，用200μl的最大体积，并用含有PBS注射器更换注射器。关键的一步:慢慢进行注射，并采取预防措施，以避免让静脉以外的解决方案。 </li></ol></li><li>通过点击的Z-Stack和时间流逝按钮来抑制它们，然后点击录制按钮开始采集的Z堆叠延时成像。 </li><li>如果其它荧光葡聚糖， 即 10 kDa的葡聚糖-荧光素异硫氰酸酯（FITC），或蛋白质， 即 ，VEGFA 165，已事先准备，图像采集在= 0分钟开始开始后注入它们。 <ol><li>在尾静脉导管除去，用PBS注射器，用含10 kDa的葡聚糖-FITC或VEGFA 165注意不要引入任何气泡进入线注射器替换。 </li><li>通过尾静脉导管缓缓注入注射到小鼠和替换用含有PBS中的注射器的注射器。 </li></ol></li><li>每30-45分钟，慢慢注入50μl的PBS或生理盐水，以保持动物的水合。 </li></ol> 8.安乐死<ol><li>在图像采集的终止，安乐死的动物。 <ol><li>增加异氟烷％至5％。 </li><li>保持5％异氟醚下的动物，直到30秒它停止呼吸，从舞台上拆下后的动物。 </li><li>执行颈椎脱位，以确保完全安乐死。 </li></ol></li></ol> 9.图像处理<ol><li>获得在16位TIFF文件的图像在每个时间点每个单独的信道，并依次保存。 </li><li>执行频谱重叠（ 即绿色荧光蛋白和CFP），消除XY漂移和电影的使用ImageJ的中9条设立的方法时间推移序列产生的分离。进行高分辨率图像13的三维表面重建。 </li></ol>

<ol>
	<li>Gerlowski, L. E., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+permeability+of+normal+and+neoplastic+tissues.">Microvascular permeability of normal and neoplastic tissues.</a> Microvasc Res. 31 (3), 288-305 (1986).</li><li>Wang, H. -. L., Lai, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Evans+blue+quantitation+in+limited+rat+tissue+samples.">Optimization of Evans blue quantitation in limited rat tissue samples.</a> Sci Rep. 4, (2014).</li><li>Dvorak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rous-Whipple+Award+Lecture.+How+tumors+make+bad+blood+vessels+and+stroma.">Rous-Whipple Award Lecture. How tumors make bad blood vessels and stroma.</a> Am J Pathol. 162 (6), 1747-1757 (2003).</li><li>Nagy, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+properties+of+tumor+surrogate+blood+vessels+induced+by+VEGF-A.">Permeability properties of tumor surrogate blood vessels induced by VEGF-A.</a> Lab Invest. 86 (8), 767-780 (2006).</li><li>Egawa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+analysis+of+vascular+permeability+in+mice+using+two-photon+microscopy.">Intravital analysis of vascular permeability in mice using two-photon microscopy.</a> Sci Rep. 3, (2013).</li><li>Yuan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+a+human+tumor+xenograft:+molecular+size+dependence+and+cutoff+size.">Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size.</a> Cancer Res. 55 (17), 3752-3756 (1995).</li><li>Dellian, M., Yuan, F., Trubetskoy, V. S., Torchilin, V. P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+a+human+tumour+xenograft:+molecular+charge+dependence.">Vascular permeability in a human tumour xenograft: molecular charge dependence.</a> Br J Cancer. 82 (9), 1513-1518 (2000).</li><li>Entenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setup+and+use+of+a+two-laser+multiphoton+microscope+for+multichannel+intravital+fluorescence+imaging.">Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging.</a> Nat Protocols. 6 (10), 1500-1520 (2011).</li><li>Entenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor+cell+movement+in+vivo.">Imaging tumor cell movement in vivo.</a> Curr Protoc Cell Biol. Chapter. 19, (2013).</li><li>Patsialou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+multiphoton+imaging+reveals+multicellular+streaming+as+a+crucial+component+of+in+vivo.+cell+migration+in+human+breast+tumors.">Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo. cell migration in human breast tumors.</a> Intravital. 2 (2), e25294 (2013).</li><li>Gligorijevic, B., Bergman, A., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+classification+links+tumor+microenvironments+with+tumor+cell+phenotype.">Multiparametric classification links tumor microenvironments with tumor cell phenotype.</a> PLoS Biol. 12 (11), e1001995 (2014).</li><li>Roussos, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mena+invasive+(MenaINV)+promotes+multicellular+streaming+motility+and+transendothelial+migration+in+a+mouse+model+of+breast+cancer.">Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.</a> J Cell Sci. 13, 2120-2131 (2011).</li><li>Harney, A. S., et al. <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+Imaging+Reveals+Local,+Transient+Vascular+Permeability,+and+Tumor+Cell+Intravasation+Stimulated+by+TIE2hi+Macrophage-Derived+VEGFA).">Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA).</a> Cancer Discov. 5 (9), 932-943 (2015).</li><li>Robinson, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+of+metastasis+in+human+breast+carcinoma:+a+potential+prognostic+marker+linked+to+hematogenous+dissemination.">Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination.</a> Clin Cancer Res. 15, 2433-2441 (2009).</li><li>Sasmono, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ovchinnikov, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Gal4-dependent+transgenes+in+cells+of+the+mononuclear+phagocyte+system+labeled+with+enhanced+cyan+fluorescent+protein+using+Csf1r-Gal4VP16/UAS-ECFP+double-transgenic+mice.">Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice.</a> J Leukcocyte Biol. 83 (2), 430-433 (2008).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</li><li>Nakasone, E. S., Askautrud, H. A., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+drug+responses+in+the+tumor+microenvironment+in+mouse+models+of+breast+cancer.">Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.</a> J Vis Exp. (73), (2013).</li><li>Weis, S., Cui, J., Barnes, L., Cheresh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+barrier+disruption+by+VEGF-mediated+Src+activity+potentiates+tumor+cell+extravasation+and+metastasis.">Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis.</a> J Cell Biol. 167 (2), 223-229 (2004).</li><li>Wyckoff, J., Gligorijevic, B., Entenberg, D., Segall, J., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Multiphoton+Imaging+of+Tumors.">High-Resolution Multiphoton Imaging of Tumors.</a> In Vivo. Cold Spring Harb Protoc. (10), (2011).</li><li>Lathia, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo.+evidence+for+tumor+propagation+by+glioblastoma+cancer+stem+cells.">Direct in vivo. evidence for tumor propagation by glioblastoma cancer stem cells.</a> PLoS One. 6 (9), e24807 (2011).</li><li>Nakasone, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer Cell. 21 (4), 488-503 (2012).</li><li>Dreher, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability,+accumulation,+and+penetration+of+macromolecular+drug+carriers.">Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers.</a> J Natl Cancer Inst. 98 (5), 335-344 (2006).</li><li>Wyckoff, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67 (6), 2649-2656 (2007).</li><li>Chittajallu, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo.+cell-cycle+profiling+in+xenograft+tumors+by+quantitative+intravital+microscopy.">In vivo. cell-cycle profiling in xenograft tumors by quantitative intravital microscopy.</a> Nat Methods. 12 (6), 577-585 (2015).</li><li>Kedrin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat Methods. 5 (12), 1019-1021 (2008).</li><li>Yano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial-temporal+FUCCI+imaging+of+each+cell+in+a+tumor+demonstrates+locational+dependence+of+cell+cycle+dynamics+and+chemoresponsiveness.">Spatial-temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness.</a> Cell Cycle. 13 (13), 2110-2119 (2014).</li><li>Yang, M., Jiang, P., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+subcellular+multicolor+imaging+of+tumor-host+interaction+and+drug+response+in+real+time.">Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time.</a> Cancer Res. 67 (11), 5195-5200 (2007).</li><li>Manning, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+reveals+conversion+between+distinct+tumor+vascular+morphologies+and+localized+vascular+response+to+Sunitinib.">Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib.</a> Intravital. 2 (1), 24790 (2013).</li><li>Alexander, S., Koehl, G. E., Hirschberg, M., Geissler, E. K., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cancer+growth+and+invasion:+a+modified+skin-fold+chamber+model.">Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.</a> Histochem Cell Biol. 130 (6), 1147-1154 (2008).</li><li>Brown, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo.+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo. measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nat Med. 7 (7), 864-868 (2001).</li></ol>

作者什么都没有透露。

在肿瘤微环境自发发生的细胞相互作用可以导致肿瘤细胞运动和血管内的变化。活肿瘤组织的高分辨率活体成像允许多细胞动力学，可以是高瞬态10,13,24的可视化。终点与分立时间点获取的可提供的在肿瘤微环境的过程的分子机制的基本信息体内测定或时间推移的图像。活体成像的研究已经被用于研究在肿瘤微环境中发生的数天的过程，生成对血管生成，肿瘤细胞迁移和响应...

从初级乳腺肿瘤的肿瘤细胞的传播已经显示出不仅涉及肿瘤细胞，但承载间质细胞包括巨噬细胞和内皮细胞。此外，肿瘤血管异常与通透性增加1。因此，确定如何肿瘤细胞，巨噬细胞和内皮细胞相互作用介导的原发肿瘤微环境的血管通透性和肿瘤细胞血管内为理解转移重要。理解血管通透性，肿瘤细胞血管内和在肿瘤微环境的多细胞相互作用的基本信令机制的动力学可提供在抗癌疗法的开发和管理的关键信息。 研究体内肿瘤血管通透性的主要手段一直是血管外染料的测量，如伊文思蓝2，高分子量的葡聚糖（155 kDa）的染料注射后3和荧光团或放射性缀合的蛋白质（包括白蛋白）4在固定的时间点。在显微镜的进步，动物模型和荧光染料已通过活体显微镜5启用细胞过程和在活体动物血管通透性的可视化。 活的动物成像采集静态图像，或短的时间推移的序列在数个时间点不允许在肿瘤微环境6,7-动态事件的完整的理解。实际上，静态图像采集在24小时的过程中显示，肿瘤血管是漏水，但血管通透性的动力学没有观察到6。因此，延长的持续时间推移成像长达12小时捕获在肿瘤微环境动态事件的动力学。 本协议描述使用较长时间推移multiphot的关于活体显微镜来研究在肿瘤微环境动态多细胞事件。在肿瘤微环境多种细胞类型标记有可注射的染料或通过使用表达荧光蛋白的转基因动物。使用尾静脉导管的血管的染料或蛋白质可成像开始之后被注入以获得在肿瘤微环境的多细胞事件的动力学数据。活细胞成像的乳腺肿瘤是通过手术准备一个皮瓣露出。图像被获取用于使用配备有多个光电倍增管（PMT）探测器8多光子显微镜长达12小时。通过使用适当的过滤器，减法算法使4 PMT检测器获取在肿瘤微环境同时9 5的荧光信号。高分辨率多光子活体显微镜捕获在肿瘤微环境肿瘤 - 基质相互作用的单细胞分辨率成像，从而导致更好ü血管通透性和肿瘤细胞血管内10-13的nderstanding。具体地，扩展的活体成像显露高度局部，瞬态血管通透性，在肿瘤细胞，巨噬细胞和内皮细胞之间的相互作用的位点选择性地发生的事件（定义为转移，TMEM 14的肿瘤微环境）13。此外，肿瘤细胞血管内仅在TMEM发生和与血管通透性13被空间和时间上相关。这些事件的动态单细胞分辨率是通过在肿瘤微环境中使用荧光标记的细胞的延长的时间推移多光子显微镜的成为可能。

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">155 kDa dextran-tetramethylrhodamine isothiocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">T1287</td>
			<td style="width:167px;">reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">70 kDa dextran-Texas Red</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">D-1830</td>
			<td>reconstitute at 10 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">10 kDa dextran-fluorescein isothyocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">FD10S</td>
			<td>reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Qdot 705 ITK Amino (PEG) Quantum Dots</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">Q21561MP</td>
			<td>Dilute 25 uL in 175 uL of 1 X PBS for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MMTV-PyMT mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">2374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">26051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-EGFP mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">1 x PBS</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Isoethesia (isoflurane)</td>
			<td style="width:95px;">Henry Schein Animal Health</td>
			<td align="right" style="width:128px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen</td>
			<td style="width:95px;">AirTech</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1 mL syringe, tuberculin slip tip</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">30G x 1 (0.3 mm X 25 mm) needle</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">305128</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Polyethylene micro medical tubing&#160;</td>
			<td style="width:95px;">Scientific Commodities Inc</td>
			<td style="width:128px;">BB31695-PE/1</td>
			<td>0.28 mm I.D. X 0.64 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope coverglass</td>
			<td style="width:95px;">Corning</td>
			<td style="width:128px;">2980-225</td>
			<td>thickness 1.5, 22 X 50 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MouseOx oximeter, software and sensors</td>
			<td style="width:95px;">STARR Life Sciences</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Laboratory tape</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:128px;">159015R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">soft rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8514K62</td>
			<td>Ultra-Soft Polyurethane Film, 3/16&#8221; Thick, 12&#34; x 12&#34;, 40 Oo Durometer, Plain Back</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">hard rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8568K615</td>
			<td>High-Strength Neoprene Rubber Sheet 1/4&#34; Thick, 12&#34; X 12&#34;, 50A Durometer,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope</td>
			<td style="width:95px;">Olympus</td>
			<td style="width:128px;"></td>
			<td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">7-Punch set</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td style="width:128px;">3429A12</td>
			<td>, 1/4&#34; to 1&#34; Hole Diameter, for Hammer-Driven Hole Punch,&#160;</td>
		</tr>
	</tbody>
</table>

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.

延长的时间推移活体显微镜使在肿瘤微环境的多细胞过程的单细胞分辨率的成像。通过荧光标记的肿瘤细胞，巨噬细胞，血管空间和可视化使用二次谐波生成信号的胶原纤维网络，在肿瘤微环境多个隔间成像期间被同时跟踪。标记的荧光蛋白的肿瘤细胞可以在转基因小鼠如已在MMTV-PyMT-Dendra2小鼠已经完成，用标记与Dendra2（ 图1A）的乳腺肿瘤细胞中产生。通过?...

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

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

这个协议描述了使用多光子显微术的实时在单细胞分辨率来执行多细胞相互作用的延长时间推移成像，体内 。

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

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

extended-time-lapse-intravital-imaging-real-time-multicellular

Research

JoVE Journal

Medicine

8.0K 次观看.  Albert Einstein College of Medicine. 这个协议描述了使用多光子显微术的实时在单细胞分辨率来执行多细胞相互作用的延长时间推移成像，体内 。 

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

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

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 excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

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 ...

科研

医学

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.

使用实时活体成像在肿瘤纳米粒子吸收的评价

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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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

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

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

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

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

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

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...