作者感谢杰里米CM张志贤和S.瑞安奥利弗的贡献和Jolanta Kilarska在图像处理有帮助。我们感谢在洛桑联邦理工学院的BIOP核心设施与双光子显微镜的支持这项工作是由欧洲研究委员会（DC-淋巴，206653-2），欧洲框架项目7（AngioScaff），瑞士国家科学基金会（31-135756），和健康的美国国家研究院​​的赞助支持部分（NIH）/美国国立卫生研究院心脏，肺和血液研究所（NHLBI）（RO1 HL096539）。此外，从罗伯特·温纳奖（瑞士癌症联盟）资金允许购买在这项研究中所使用的徕卡立体显微镜。资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有任何作用。

对动物进行的所有程序均严格按照瑞士动物保护法，动物保护条例及动物实验的条例。我们确认，我们的机构动物护理和使用委员会（IACUC），命名委员会去监督DE L&#39;行政法院去沃州（许可证编号:2687），特别是批准了这项研究。 1，肿瘤的耳接种<ol><li>培养B16-F10-GFP的黑色素瘤细胞中使用的DMEM +10％FBS媒体10cm的培养皿中。 </li><li>当细胞80-90％汇合时，用PBS冲洗，并通过胰蛋白酶消化分离它们。降速细胞在1500×g离心5分钟，在4℃，重悬沉淀于1毫升林格氏溶液，并将其转移到1.5ml的Eppendorf管中。再次旋转，并删除所有上清液，除了一层薄薄的介质是仅仅停留在小球上面。重悬细胞结了厚厚的细胞浆。 </li><li>麻醉鼠标KET的混合物胺/ dorbene [美托咪定]（75mg/kg-1mg/kg），并确认该动物是充分通过执行一个温柔的脚趾捏麻醉。增加异氟醚浓度的情况下运动步长为0.1％观察（ 如爪子撤出）。在整个过程中保持鼠标在直肠热敏电阻控制的加热垫（37°C）和保护其眼睛以适当的眼药膏。 </li><li>准备一个汉密尔顿注射器（33号针头，将10毫升注射器体积）。用针除去的尖端帽，并加载20微升细胞浆液的针尖上腔室的顶部。直到5毫升浆注射器里面装回拉柱塞。从尖端室去除多余的浆液，并关闭尖帽。 </li><li>使用粘合带，固定在耳朵上的食指的尖端处的近侧边缘。在一个45°角，慢慢地插入背真皮和软骨之间的汉密尔顿注射器针头。一旦进入，穿透耳近端至远端约2-3毫米。 <b转/&gt;注:细胞注射的过程应该由受过培训和认证的技术人员来完成。此外，这一过程必须缓慢进行，并在序列:第一针应水平放置在耳后皮肤并插入慢慢进入耳真皮。一旦才把针头端是在皮肤上，所述柱塞应按压。按下柱塞将启动细胞接种在皮肤上。在针穿过耳真皮中，软骨层并进入执行实验的人的皮肤的两个层的异常情况下，造成的experimentator手指疼痛应被视为以惊人的符号，并导致立即缩回从experimentator皮肤的针。在这种情况下，柱塞不应该被压制和皮肤应该用酒精进行消毒。可替代地，无生命的物体，而不是手指可以用来稳定耳。 </li><li>注3微升细胞浆，慢慢地缩回从耳朵针。让我们在7-9天的肿瘤细胞形成实体瘤，并按照肿瘤的生长通过使用荧光显微镜。 </li><li>另外，遵循早期步骤与组织基质肿瘤细胞相互作用的，适用50微升100,000肿瘤细胞悬浮在林格的揭露和染色真皮耳（3.3步后）解决方案。 </li></ol> 2，耳手术<ol><li>实验前三天，剃了头鼠标，脱毛在头部和耳朵（10-15秒）的头发和用清水冲洗干净。 </li><li>麻醉鼠标加湿氧气和异氟醚（3-4％感应，1-2％的维护）的混合物，并确认该动物是充分通过执行一个温柔的脚趾捏麻醉。增加异氟醚浓度的情况下运动步长为0.1％观察（如爪子撤出）。在整个过程中保持鼠标在直肠热敏电阻控制的加热垫（37°C）和保护它的眼睛与合适的眼药膏。 </li><li>建造出来的8粘玻璃病理切片的平台。打开背面的鼠标，轻轻放置玻片堆栈中的荷瘤耳。使用的粘接带小条纹注视耳朵的前部和后部边缘到堆栈中。 </li><li>切割沿使用手术刀鼠标耳廓的耳轮的耳腹侧皮肤。具有弯曲镊子的帮助下，轻轻地撕掉腹侧真皮和从背侧真皮其中肿瘤接种软骨。拉断腹侧皮肤和软骨弯钳，露出背部真皮。注:如果在耳朵的主要血管被切断或血液循环不在15分钟内恢复正常流量时，耳不能用于成像。总是用林格氏缓冲液保持在打开的耳朵皮肤湿润并用盖玻片保护的湿度。 </li><li>但如果是持续性出血形式肿瘤血管中，加入100微升止血凝血酶（5单位/毫升）在林格氏缓冲液（102 mM氯化钠，5mM的氯化钾，2mM的氯化钙 ，28毫乳酸钠）在耳朵上进行5分钟的顶部。 </li><li>用大约5毫升林格氏缓冲液洗两次耳朵并删除多余的液体用无菌湿巾。立即进行下一个步骤（不要让开干耳在任何点！）。 </li></ol> 3，免疫荧光染色使用林格氏缓冲液补充了人血清（1:10），小鼠多克隆第二抗体对人IgG（1:50），和125国际单位/毫升（2.5毫克/毫升）的抑肽酶（封闭缓冲液）对所有的染色步骤。抑肽酶抑制纤溶酶促进凝血，限制手术后可能发生的初始​​出血。 <ol><li>折叠的耳部与开放真皮成eminentia鼻甲和干燥外未开封的耳真皮用无菌纸巾。通过将0.5微升的手术胶水的前部和后部手背固定耳载玻片在堆栈上升边缘。然后，轻轻压平背真皮耳到玻璃上。 </li><li>适用于初级抗体，以10微克/毫升中的100微升封闭缓冲液中的露耳的总体积的浓度靶向细胞外基质分子。覆盖，以防止染色液干燥在耳朵边盖玻片耳。孵育15分钟。用大约5毫升林格氏缓冲液洗两次耳朵。 </li><li>在以100微升封闭缓冲液中的露耳的总体积10微克/毫升的浓度采用适当的二级抗体或链霉亲和结合物（荧光团具有高的激发波长，例如594纳米或647纳米有利于活体成像）。盖上盖玻片耳朵孵育15分钟。用大约5毫升林格氏缓冲液洗两次耳朵。 </li></ol>血源性激活的脾细胞4。 原位肿瘤细胞的相互作用<ol><li> inoculatio后7天对同类系小鼠背部皮肤GFP-B16-F10黑素瘤N，安乐死的动物，并收集脾脏。通过一个70微米的细胞过滤分离脾细胞与脾的机械破坏。 </li><li>标签的脾细胞在37℃下8分钟，使用红CMTPX细胞的细胞质染色（1μM在PBS中），在15毫升洗涤4次，在4℃，并立即用200μl无血清培养基注入到尾静脉对小鼠的耳朵接种B16-F10-GFP7天更早。 </li></ol> 5，活体成像通过使用体视显微镜<ol><li>对于短期成像（最多2小时），添加含有140毫抗坏血酸钠，10mM的HEPES，4mM的氯化钾和5mM的CaCl 2中 ，在pH为7.5（抗坏血酸盐林格氏缓冲最终新鲜配制的无菌抗坏血酸盐林格氏缓冲渗透压320毫渗量）上的固定耳朵的上方。盖上盖玻片耳朵，并通过使用荧光显微镜用2X透镜开始成像。 </li><li>对于长期成像（超过2小时），将针的插座（连接到含有抗坏血酸盐林格氏缓冲储存器）的盖玻片下，约0.5厘米的距离的耳部。使用蠕动泵以1微升/分钟的速度在盖玻片下，不断提供抗坏血酸林格氏缓冲液到腔室。 </li><li>改变1X透镜（工作距离6厘米），以2X透镜（工作距离为2厘米）。打开采集软件，并配置了荧光显微镜的设置。在相机设置中选择12位作为图像的颜色深度和从0％（最低）调整灰度值的摄像头范围为5.1％（最大值），并设置伽玛校正2。 <ol><li>置采集的增益1（最小），荧光强度1000（最大），放大倍数设定为280X-320X和调节曝光时间，避免过度或曝光（曝光时间应小于1秒）。视摄像视场（通常为10至20），用于采集周期的时间间隔的数目应被设置为之间1至2分钟。缩短的时间可能会导致漂白和光毒性。 </li></ol></li><li>选择包含肿瘤细胞以及染色ECM蛋白（例如图3）几个字段。使用电动载物台，取得相应的荧光通道（如绿色荧光蛋白和荧光团647在图3）随时间（ 例如每2分钟）的图像。 </li><li>实验结束后，根据机构的动物安乐死的准则鼠标。在这种情况下，在实验结束时，通过颈脱位接着放血（心脏内灌注）安乐死麻醉小鼠。 </li></ol> 6，活体成像通过使用多光子显微镜<ol><li>使用硅润滑脂，建立一个圆形壁约2厘米直径和耳朵周围2-3毫米的高度，开始于耳的基部。确保没有泄漏点。充满抗坏血酸林格氏缓冲圈。 </li><li>将鼠标在舞台上，并连接在加热垫（37°C）。 </li><li>开放获取软件和配置多光子显微镜的设置。 </li><li>选择多达四个包含的肿瘤细胞和染色的细胞外基质蛋白（如在图3中）的不同字段。在这个例子中，调整的Ti-萨费尔激光到850 nm的GFP信号，随后的单光子647为荧光团647染色和可视化纤维状胶原与SHG。获得适当的荧光通道（如GFP和荧光团647在图3中）的图像和二次谐波产生随着时间的推移， 例如每2分钟，用水浸没HCX APO 20X与1.00 NA透镜和2毫米的工作距离。 </li><li>实验结束后，安乐死麻醉小鼠颈椎脱臼接着放血（心内灌注）。 </li></ol>

<ol>
	<li>Martinez-Corral, I., 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+imaging+of+lymphatic+vessels+in+development,+wound+healing,+inflammation,+and+tumor+metastasis.">In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis.</a> Proc Natl Acad Sci. , (2012).</li><li>Snippert, H. J., 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=Intestinal+crypt+homeostasis+results+from+neutral+competition+between+symmetrically+dividing+Lgr5+stem+cells.">Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.</a> Cell. 143, 134-144 (2010).</li><li>Halin, C., Mora, J. R., Sumen, C., von Andrian, U. H. <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+imaging+of+lymphocyte+trafficking.">In vivo imaging of lymphocyte trafficking.</a> Annu. Rev. Cell Dev. Biol. 21, 581-603 (2005).</li><li>Kilarski, W., Petersson, L., Fuchs, P., Zielinski, M., Gerwins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+neovascularization+assay+for+screening+regulators+of+angiogenesis+and+assessing+their+effects+on+pre-existing+vessels.">An in vivo neovascularization assay for screening regulators of angiogenesis and assessing their effects on pre-existing vessels.</a> Angiogenesis. 15, 643-655 (2012).</li><li>Kilarski, W. W., 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+Immunofluorescence+for+Visualizing+the+Microcirculatory+and+Immune+Microenvironments+in+the+Mouse+Ear+Dermis.">Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis.</a> PLoS ONE. 8, (2013).</li><li>Steinbauer, M., 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=GFP-transfected+tumor+cells+are+useful+in+examining+early+metastasis+in+vivo,+but+immune+reaction+precludes+long-term+tumor+development+studies+in+immunocompetent+mice.">GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.</a> Clin. Exp. Metastasis. 20, 135-141 (2003).</li><li>Ohashi, T., Kiehart, D. P., Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+elasticity+of+the+fibronectin+matrix+in+living+cell+culture+visualized+by+fibronectin-green+fluorescent+protein.">Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein.</a> Proc Natl Acad Sci U S A. 96, 2153-2158 (1999).</li><li>Egeblad, M., 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=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis. Models Mech. 1, 155-167 (2008).</li><li>Tal, O., 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=DC+mobilization+from+the+skin+requires+docking+to+immobilized+CCL21+on+lymphatic+endothelium+and+intralymphatic+crawling.">DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling.</a> J. Exp. Med. 208, 2141-2153 (2011).</li><li>Roos, A., Trouw, L. A., Ioan-Facsinay, A., Daha, M. R., Verbeek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Life Sciences. , 1-14 (2005).</li><li>Kilarski, W. W., Samolov, B., Petersson, L., Kvanta, A., Gerwins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+regulation+of+blood+vessel+growth+during+tissue+vascularization.">Biomechanical regulation of blood vessel growth during tissue vascularization.</a> Nat Med. 15, 657-664 (2009).</li><li>Sahai, E., 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=Simultaneous+imaging+of+GFP,+CFP+and+collagen+in+tumors+in+vivo+using+multiphoton+microscopy.">Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy.</a> BMC Biotechnol. 5 (1), 14 (2005).</li><li>Wyckoff, J. B., Jones, J. G., Condeelis, J. S., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+step+in+metastasis:+In+vivo+analysis+of+intravasation+at+the+primary+tumor.">A critical step in metastasis: In vivo analysis of intravasation at the primary tumor.</a> Cancer Res. 60, 2504-2511 (2000).</li><li>Pinner, S., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+amoeboid+cancer+cell+motility+in+vivo.">Imaging amoeboid cancer cell motility in vivo.</a> J. Microsc. 231, 441-445 (2008).</li><li>L&#228;mmermann, 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453, 51-55 (2008).</li><li>Padera, T. P., Stoll, B. R., So, P. T., 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=Conventional+and+high-speed+intravital+multiphoton+laser+scanning+microscopy+of+microvasculature,+lymphatics,+and+leukocyte-endothelial+interactions.">Conventional and high-speed intravital multiphoton laser scanning microscopy of microvasculature, lymphatics, and leukocyte-endothelial interactions.</a> Mol. Imaging. 1, 9-15 (2002).</li><li>Giampieri, 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=Localized+and+reversible+TGFbeta+signalling+switches+breast+cancer+cells+from+cohesive+to+single+cell+motility.">Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility.</a> Nat. Cell Biol. 11, 1287-1296 (2009).</li><li>Weber, M., 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=Interstitial+dendritic+cell+guidance+by+haptotactic+chemokine+gradients.">Interstitial dendritic cell guidance by haptotactic chemokine gradients.</a> Science. 339, 328-332 (2013).</li><li>Midwood, K. S., Orend, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+tenascin-C+in+tissue+injury+and+tumorigenesis.">The role of tenascin-C in tissue injury and tumorigenesis.</a> Journal of cell communication and signaling. 3, 287-310 (2009).</li><li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biological Confocal Microscopy. , 340-341 (2006).</li><li>Sund, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stroma+derived+biomarkers+in+cancer.">Tumor stroma derived biomarkers in cancer.</a> Cancer Metastasis Rev. 28, 177-183 (2009).</li><li>Mueller, M. M., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+foes+-+bipolar+effects+of+the+tumour+stroma+in+cancer.">Friends or foes - bipolar effects of the tumour stroma in cancer.</a> Nat Rev Cancer. 4, 839-849 (2004).</li><li>Brown, E., 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=Dynamic+imaging+of+collagen+and+its+modulation+in+tumors+in+vivo+using+second-harmonic+generation.">Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.</a> Nat. Med. 9, 796-800 (2003).</li><li>Rice, J. J., Gerwins, P., Kilarski, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Angiogenesis:+Perspectives+from+Antiangiogenic+Tumor+Therapies.">Mechanisms of Angiogenesis: Perspectives from Antiangiogenic Tumor Therapies.</a> Current Angiogenesis. 1, 139-147 (2012).</li><li>Patterson, G. H., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+in+two-photon+excitation+microscopy.">Photobleaching in two-photon excitation microscopy.</a> Biophysical journal. 78, 2159-2162 (2000).</li><li>Jakobsson, L., 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=Heparan+sulfate+in+trans+potentiates+VEGFR-mediated+angiogenesis.">Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis.</a> Dev Cell. 10, 625-634 (2006).</li><li>Kilarski, W. W., 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=Optimization+and+regeneration+kinetics+of+lymphatic-specific+photodynamic+therapy+in+the+mouse+dermis.">Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.</a> Angiogenesis. , (2013).</li></ol>

作者什么都没有透露。

意义在这里，我们提出一种新的活体显微镜方法，使高分辨率和动态不同组织微环境的组件，包括纤维状以及网格状的基质蛋白的可视化。该方法具有过电流的活体成像技术几个优点:（ⅰ）活体荧光成像已被用于在微循环研究， 例如 ，以跟踪从血液或淋巴管进入溶质泄漏，但一直没有结合免疫染色。 （ⅱ）利用转基因报告小鼠的允许要被成像的特定细胞类型，但要求它们的可用性（或大量的努力以创建新的），并限制了该相互作用可以研究的细胞类型的数量。 （ⅲ）细胞外基质可在体内使用的二次谐波生成进行成像，但是这种技术只能检测纤维状胶原，留下了大量重要的细胞外成分如基底膜，纤连蛋白，tenascins，生长因子，趋化因子和组织粘多糖出目前的研究遥不可及。我们的方法克服了这些限制，并允许掺入标准成像技术，并进一步结合immunostainings为其他类型的细胞，组织结构，硫酸肝素的存款结合生长因子（ 例如血管内皮生长因子26）和趋化因子（CCL21 5,18和图2D ）或胞外基质蛋白，而同时跟踪血液和/或淋巴流。 限制活体中频的落射荧光成像仅限于薄皮瓣，剥夺厚皮下组织（脂肪组织）的。而我们发现，背耳真皮是最优的耳部皮肤，落射荧光与活体的相对无害的外科论述中频技术不限于耳真皮和可能被应用到如脚趾或新生小鼠的背部皮肤裸露的皮肤。快速抗体染色（15分钟）在免疫荧光IF是不依赖于被动扩散，但需要有功能的淋巴引流和间质液流动，因此，如果淋巴管被封闭27无染色可以观察到。在与该行，淋巴管染色更强然后渗漏血液的血管（血管损伤，动脉）5。背侧和腹侧耳皮瓣的分离是一个温和的外科手术，但它会导致伤害一些毛细管和细胞死亡的各种组织细胞。这可能是一个问题，当一个需要探讨完全完整的组织， 例如在看药物对细胞存活的轻微影响。也抗氧化剂的应用是用于保护皮肤免受光毒性和光漂白排除了使用活体免疫荧光技术的研究，例如在组织的氧化应激或一氧化氮BIOLOG年。 最后，与免疫复合物致盲的居民组织的巨噬细胞可激活这些细胞，并影响如组织免疫反应和树突状细胞活化的研究。 修改和故障排除为了研究组织的氧化应激或一氧化氮的生物，抗坏血酸，必须更换带不能与实验输出干扰其他组织相容的介质。类似地，应尽量避免挡住Fc受体上真皮的免疫细胞，如果研究的目的是函数和活化的组织的免疫反应和树突状细胞的活化和迁移。这可以通过使用二次抗体与Fc片段裂解（的F（ab）2抗体片段），或使用生物素化的抗体和链亲和素的荧光作为检测试剂来完成。 未来的应用我们提出了一个新颖而独特的内重要中频技术细胞外基质中移植皮肤肿瘤中使用双光子显微镜SHG-检测纤维状和成熟的胶原相结合的图像非纤维状部件。一种利用多光子（或共焦）显微镜落射荧光过成像的优点为z-堆积的可能性，而由于空间的共定位外基质内发生的事件， 例如 ，肿瘤血管内进入淋巴管或血管，其中在的情况下标准荧光显微镜可以从入侵的细胞形态变化只能推断。该技术具有更加宽广的潜力。例如，我们已经使用了活体中频探讨淋巴管阻塞淋巴管特异性光动力疗法27的机制。另外的潜在这种方法的应用包括，但没有在移植排斥或血液和淋巴方法炎症，机制不限于皮肤免疫调查<html>肿瘤发生过程中ATIC血管的生长。 在该过程的关键步骤有对维持生理组织环境影响的关键最重要的一步是腹侧皮肤和软骨背侧真皮的手术分离。切断或阻塞的主要动脉或静脉会导致出血过多和局部组织缺氧，这将影响到治疗和细胞运动8细胞反应。固定手术胶耳应谨慎做作为溢出的胶水暴露组织会造成永久性的伤害组织由血管闭塞。鼠标的温度必须控制并保持在37℃。此外，加湿的氧气需要被用于异氟烷麻醉，使眼睛和肺停留在实验过程中适当地加湿。此外，抗坏血酸钠，必须新鲜配制，使用前其PH值检查。 ntent"&gt;总之，这个活体免疫的桥梁生理功能与在小鼠皮肤复杂的细胞活动分子成像的实时，局部测量。此外，这种方法具有很大的潜力，因为它可以很容易地应用到如研究后发育血液和淋巴血管生成，或由各种病原体可视化皮肤感染的早期阶段。凭借其灵活性和高通量的潜在机制，这种活体技术可显著促进生物学的多个领域。

炎症性，转移性和基质重塑过程的活体成像一直是一个重要的研究领域，并激发创造，表达荧光蛋白1,2众多记者的转基因小鼠模型。由于失焦荧光信号，从而降低图像质量和限制穿过组织轻微渗透，成像厚的组织是可能的，只有用共聚焦或多光子扫描显微镜3。使用更快，更昂贵和复杂的荧光显微镜可以仅与近二维组织如鸡绒毛膜尿囊膜4或小鼠耳真皮5。大多数成像系统采取的转基因小鼠的优势表达各种荧光蛋白的细胞类型特异性的方式。尽管这些蛋白报价弱势光毒性，它们诱导免疫应答6。此外，也难以特定细胞亚群之间切换或标记IR基或激活状态的这种变化需要新的遗传模型的制备。此外，作为荧光蛋白表达通常限于胞内区室，它通常是不可能像基底膜蛋白或组织趋化因子存款7图像胞外结构。相反，具有抗细胞外抗原的间接标记提供了灵活性，几乎所有的细胞类型或矩阵特定组件8,9。然而，这种标记方法的主要缺点是与免疫毒性通过抗原-抗体免疫复合物，可以触发补体系统依赖的细胞毒性和细胞与细胞外结构10介导的吞噬作用相关联。 肿瘤微环境中的炎症或创伤愈合过程中的细胞外基质的正常组织中，而且还发生实质性的重塑。肿瘤微环境中的基质变硬可以促销TE肿瘤的生长和侵袭，由于压力引起的信号传导机制及血液和淋巴管11的原因重塑。然而，基质蛋白的实时成像仅限于能够由二次谐波生成的使用多光子显微术来检测纤维状胶原。最近我们已经公布，最大限度地减少照片和免疫毒性损伤成像细胞和组织结构5的风险了一种新的活体成像的方法。在既定的成像技术，其中的单轮的连续活体可视化是在范围为30分钟至2小时12-17相比，我们的活体免疫荧光（IF）技术允许12小时（长期8）成像。值得注意的是，成像时间被限制到由于在我们的动物协议定义的限制12小时但没有任何技术反迹象表明，它不能长时间是非常重要的，如果动物的健康至关重要的参数，如血压和心脏率控制8。此外，通过使用自动化荧光显微镜，我们能够在一个单一实验中，收集从多个字段的图像，并观察到发生在由功能血液和淋巴管支持皮肤的生理上下文罕见的免疫和重塑的过程。由于显微立体透镜具有相对大的2厘米，工作距离和相对于双光子显微光学不需要汽化水浸泡，进行只在荧光立体显微镜长期成像。活体如果允许正常皮肤的任何细胞外基质成分的免疫标记和检测。这种技术的设计是基于使用免疫活细胞和手术暴露小鼠真皮组织基质元素，而不会造成有害的免疫毒性效应5的创新理念。在外科手术过程本身是安全的，真皮脉管系统，因为它只是依靠两个皮肤层中的独立支配，由血液输送自主和独立的淋巴环流倒掉耳朵的分离。我们的实验装置允许的范围内比至少为12小时，包括血液和淋巴管和伤口愈合过程之间白细胞运输重要病理生理事件的成像。在原始出版物，我们比较我们的技术，先进设备，最先进的活体成像的各个领域的标准。因此，我们通过不同类型的白细胞，包括进入收集，而不是预期的初始淋巴管15的免疫细胞的独特的可视化观察血管外渗和淋巴血管内的事件。此外，补充其他团体9,18所做的观察，我们发现， 在体内 CCL21能够形成于收集，但只有很少的初始淋巴管强，连续存款。 19基本上还未上下文用于成像癌细胞侵袭此方法。我们发现，肿瘤的纤维状基质，以产生二次谐波检测的，占据了肿瘤微环境中的不同位置较新沉积的基质腱生蛋白C的组成此外，我们描述了所引起的局部基质收缩血管重塑的例子中，长与肿瘤细胞和沿着基底膜IV型胶原的肿瘤细胞迁移的T细胞相互作用术语（12小时）成像。这些事件可以同时记录当地的生理参数，如血管PE成像rmeability和淋巴引流。

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			<td height="21" style="height:21px;width:515px;">Mice Strain</td>
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			<td height="21" style="height:21px;width:515px;">BALB/C mice (8-12 weeks )</td>
			<td style="width:159px;">Charles River&#160;</td>
			<td style="width:119px;"></td>
			<td>Orleans,France</td>
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			<td height="21" style="height:21px;width:515px;">C57/Bl6 Mice (8-12 weeks )</td>
			<td style="width:159px;">Harlan&#160;</td>
			<td style="width:119px;"></td>
			<td>Carshalton UK</td>
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			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:515px;">Cell Line&#160;</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:515px;">B16 F10 Melanoma expressing OVA-GFP&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:515px;">Anesthesia Maintenance&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:515px;">Isofluorane&#160;</td>
			<td style="width:159px;">Minrad Inc.&#160;</td>
			<td align="right" style="width:119px;">2222</td>
			<td>Minrad Inc., Buffalo, NY</td>
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			<td style="width:159px;">Rothhacher GmBH&#160;</td>
			<td style="width:119px;"></td>
			<td>Berne, Switzerland&#160;</td>
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			<td height="21" style="height:21px;width:515px;">DC Temperature Control System&#160;</td>
			<td style="width:159px;">FHC Inc&#160;</td>
			<td style="width:119px;"></td>
			<td>Bowdoin, MA</td>
		</tr>
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			<td height="21" style="height:21px;width:515px;"></td>
			<td style="width:159px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Stereomicroscope&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;">Fluorescence stereomicroscope with a motorized stage .</td>
			<td style="width:159px;">M250 FA, Leica Microsystems CMS GmbH</td>
			<td style="width:119px;"></td>
			<td>Wetzlar, Germany</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:515px;">&#160;1&#215; lens (linear system magnification from 7.5&#215; to 160&#215;)&#160;</td>
			<td style="width:159px;">Leica Microsystems CMS GmbH</td>
			<td style="width:119px;"></td>
			<td>Wetzlar, Germany</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:515px;">2&#215; lens (linear system magnification from 15.6&#215; to 320&#215;)</td>
			<td style="width:159px;">Leica Microsystems CMS GmbH</td>
			<td style="width:119px;"></td>
			<td>Wetzlar, Germany</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:515px;">DFC 350 FX camera controlled by LAS AF software&#160;</td>
			<td style="width:159px;">Leica Microsystems CMS GmbH</td>
			<td style="width:119px;"></td>
			<td>Wetzlar, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;"></td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Multiphoton Microscopy&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">LEICA SP5, two-photon multiplier, motorized stage&#160;</td>
			<td style="width:159px;">Leica Microsystems CMS GmbH</td>
			<td style="width:119px;"></td>
			<td>Wetzlar, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCX APO 20x/1.2 oil immersion&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td style="width:159px;"></td>
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			<td style="width:159px;"></td>
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			<td></td>
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			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:515px;">Ringer&#39;s buffer (102 mM NaCl, 5 mM KCl, 2 mM CaCl2, 28 mM sodium lactate)</td>
			<td>B. Braun Medical AG</td>
			<td align="right">445968</td>
			<td>Sempach, Germany</td>
		</tr>
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			<td height="21" style="height:21px;width:515px;">Aprotinin&#160;</td>
			<td>Elastin</td>
			<td>AP92</td>
			<td>Owensville, MO</td>
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			<td height="21" style="height:21px;width:515px;">Thrombin&#160;</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td>T7326-1KU&#160;</td>
			<td>Taufkirchen, Germany&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium ascorbate</td>
			<td style="width:159px;">Sigma-Aldrich&#160;&#160;</td>
			<td>11140-50G&#160;</td>
			<td>Taufkirchen, Germany&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Mouse Serum raised against human IgG&#160;</td>
			<td style="width:159px;">Abcam&#160;</td>
			<td>ab34834&#160;</td>
			<td>Cambridge, UK</td>
		</tr>
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			<td height="21" style="height:21px;width:515px;">Collagen IV&#160;</td>
			<td>Abcam</td>
			<td>AB6581</td>
			<td>Cambridge, UK</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Perlacan&#160;</td>
			<td style="width:159px;">RnD&#160;</td>
			<td>ab79465&#160;</td>
			<td>Minneapolis, MN</td>
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			<td height="21" style="height:21px;width:515px;">Tenascin C&#160;</td>
			<td style="width:159px;">RnD&#160;</td>
			<td>AF3358</td>
			<td>Minneapolis, MN</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Podoplanin</td>
			<td style="width:159px;">RnD&#160;</td>
			<td style="width:119px;">AF3244</td>
			<td>Minneapolis, MN</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">LYVE-1&#160;</td>
			<td style="width:159px;">Reliatech&#160;</td>
			<td>103-PA50&#160;</td>
			<td>Wolfenb&#252;ttel, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">CCL21</td>
			<td style="width:159px;">RnD</td>
			<td style="width:119px;">AF457</td>
			<td>Minneapolis, MN</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Streptavidin Pacific Blue&#160;</td>
			<td style="width:159px;">Invitrogen&#160;</td>
			<td>S11222</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Streptavidin Alexa 647&#160;</td>
			<td style="width:159px;">Invitrogen&#160;</td>
			<td>S21374</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Streptavidin Alexa 488&#160;</td>
			<td style="width:159px;">Invitrogen&#160;</td>
			<td style="width:119px;">S11223</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Donkey Anti Goat 594&#160;</td>
			<td style="width:159px;">Invitrogen&#160;</td>
			<td>A21113</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Donkey Anti Rabbit 594&#160;</td>
			<td style="width:159px;">Invitrogen&#160;</td>
			<td>A21207</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">CMTPX CellTracker</td>
			<td style="width:159px;">Lifetechnologies</td>
			<td style="width:119px;">C34552</td>
			<td>Carlsbad, California</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Histocryl (surgical glue)</td>
			<td style="width:159px;">Braun Aesculap</td>
			<td style="width:119px;">1050060</td>
			<td>Tuttlingen, Germany</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;"></td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Cell Culture Reagents&#160;</td>
			<td style="width:159px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM</td>
			<td>Gibco Invitrogen</td>
			<td>E15-843&#160;</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS</td>
			<td>Gibco Invitrogen</td>
			<td style="width:119px;"></td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Gibco Invitrogen</td>
			<td>25300062</td>
			<td>Grand Island, NY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">PBS&#160;</td>
			<td>Gibco Invitrogen</td>
			<td style="width:119px;"></td>
			<td>Grand Island, NY</td>
		</tr>
	</tbody>
</table>

long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy

除了是一个物理支架，以维持组织形态，细胞外基质（ECM）积极参与制定和器官的动态平衡过程中调节细胞和组织的功能。它由通过生化，生物力学和生物物理信号传导途径，如通过作用的生物活性细胞外基质蛋白的片段的释放，调节组织张力，并提供途径的细胞迁移这样做。肿瘤微环境中的细胞外基质发生实质性重构，其特征在于，降解，沉积和组织纤维状和非纤维状基质蛋白。肿瘤微环境中的基质变硬可以促进肿瘤的生长和侵袭，并造成血液和淋巴血管重塑。基质蛋白的实时成像，但是，这点是有限的，可以通过产生二次谐波的使用多光子显微术来检测纤维状胶原，而使大多数基质组分的升argely无形。在这里，我们描述了在薄耳背侧皮肤肿瘤接种程序，免疫标记细胞外基质蛋白，并用萤光和双光子显微镜在活小鼠暴露组织的活体成像的。我们的活体成像方法允许直接检测两个纤维状和非纤维状基质蛋白中的一个生长的皮肤肿瘤的上下文。我们表明所引起的局部基质收缩血管重塑的例子。我们还发现，与二次谐波生成检测出的肿瘤的纤维状基质是由新沉积的基质组分如腱生蛋白C。我们还表明T细胞相互作用的长期（12小时）成像，肿瘤细胞和肿瘤细胞在空间上不同的沿着基底膜的IV型胶原的迁移。两者合计，该方法唯一允许同时检测肿瘤细胞，它们的物理和微环境随时间的内源性组织的免疫反应，其可以省IDE的重要见解相关肿瘤进展和最终的成功或抵抗治疗的机制。

迄今为止，免疫染色在活组织通常不使用，由于形成免疫复合物，导致高背景染色和免疫毒性20。这是克服预先阻断组织巨噬细胞Fcγ受体，从而"致盲"这些细胞对后续的间接免疫染色强。作为标记为细胞外，光毒性和荧光漂白可以由组织浸没在天然抗氧化剂进行缓冲，等渗抗坏血酸（ 图1A）进 ​​行控制。由于这种方法5的我们最初的描述中，我们改善了我们的抗褪色和抗光毒技术，使漂白现在不仅可以得到抑制，但完全阻止（ 图1B）。这是通过在将耳被固定在腔室中的大量抗氧化剂（100微升）的嵌入耳完成。需要注意的是300秒不断成像时间相当于10小时的影像，当图片收集500毫秒每分钟（平均成像设置）。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51388/51388fig1highres.jpg" src="/files/ftp_upload/51388/51388fig1.jpg" /> 图1的光漂白和光毒性反应停止用抗坏血酸代替氯在林格氏缓冲液和嵌入在该溶液中加入100μl体积的露耳。 （A）组织首先用抗IV型胶原，基底膜的成分的生物素标记的抗体染色。在染色后用链亲和素-647（红）检测到，然后不断地在任一正常林格氏缓冲液（上面板）或抗坏血酸盐林格氏（下图）成像为300秒。需要注意的是300秒不断成像时间相当于10小时的影像时，会收集500毫秒每分钟（通常的成像设置）的照片。 pi的亮25％XEL强度值是绿色的。免疫荧光衰减（B）定量（50％林格主场迎战抗坏血酸林格氏0％）。值进行归一化到初始荧光。用免疫荧光显微镜采集到的图像。在比例尺 ，100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/51388fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 调查肿瘤转移细胞和肿瘤基质之间的相互作用是非常重要的了解肿瘤细胞迁移，肿瘤免疫力的方法。这是因为在肿瘤相关基质构成高达90％的肿瘤块并积极调节肿瘤生长和转移扩散21。然而，机械的深入了解，对无论是淋巴管或血管矩阵驱动肿瘤细胞的迁移缺乏22。这部分是由于这一事实，即活体即时通讯基质蛋白的老化被限制为使用双光子显微镜23的二次谐波生成纤维状胶原的成熟纤维的可视化。因此，适于将组织微环境，包括血液和淋巴管，周细胞，神经，肌肉和脂肪细胞，包括细胞外基质成分的直接可视化的可视化我们的方法。免疫染色基底膜成分，如IV型胶原或基底膜蛋白聚糖（ 图2A），然而，直接染色法对特异性细胞表面标志物， 例如 LYVE1（ 图2B和C）和podoplanin的后根据它们的形态许多结构可以区分（ 图2（c）），还允许区分初始，毛细血管淋巴管（LYVE1 +）从淋巴收藏家（LYVE1 - ）。在皮肤染色基质结合CCL21揭示这种趋化因子的存款上的淋巴收藏家IDE基底膜ntified与染色基底膜蛋白聚糖，乙酰肝素硫酸盐蛋白聚糖（ 图2D）。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51388/51388fig2highres.jpg" src="/files/ftp_upload/51388/51388fig2.jpg" /> 图中正常小鼠耳活染色2。例子。 （ 一 ）染色的基底膜，基底膜成分，描绘所有的血液和淋巴管，神经，肌肉纤维和脂肪细胞。 （二）LYVE1染色标志着初始毛细淋巴管网络。 （ 三）联合染色LYVE1和podoplanin的，一个泛淋巴管标记，描绘了最初的和收集淋巴管网络。背真皮耳可以使用传统的荧光显微镜（不含光学切片）作为耳真皮层进行成像具有脂肪细胞的数量少，否则将覆盖成像领域。 （四）CCL21的领带<html>宁（绿色）淋巴管基底膜染色串珠素（红色）。用免疫荧光显微镜采集到的图像。标尺A和B，1毫米;在C中，100μm和50μm的D中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/51388fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 最重要的是，通常不能由SHG，经典的矩阵检测方法23进行检测的结构蛋白，可以可视化。例如，我们发现，生腱蛋白C（ 图3A），这是肿瘤发生过程中表达基质蛋白，伤口愈合和炎症19沉积在肿瘤间质的不同位置比纤维胶原蛋白（ 图3B）。这个矩阵的异质性可能会影响肿瘤细胞的分布和转移（ 图3C）。 <html> T"FO:保持together.within页="总是"&gt; <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51388/51388fig3highres.jpg" src="/files/ftp_upload/51388/51388fig3.jpg" /> 图3:背真皮耳可使用多光子显微镜实时成像。瘤B16-F10的单场。肿瘤基质被标记肌腱肝炎抗体和染色用抗羊-594驴抗体检测。腱糖蛋白C（红色）的免疫网络如图A和B中检测到二次谐波产生（SGH）纤维胶原蛋白。这两个网络都使用C（合并）叠加与肿瘤细胞（青色）。标志着肌腱C新型肿瘤基质不重叠与SHG（绿色）检测纤维胶原蛋白。 44，四次均z轴部分与Z-级1.0微米的图像是在16位色深模式获得的。用双光子显微镜采集的图像。比例尺，100微米。REF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/51388fig3highres.jpg"目标="_blank"&gt;请点击这里查看该图的放大版本。 以下不同的肿瘤基质成分的肿瘤基质（ 如肌腱C和Ⅳ型胶原）的免疫标记我们可以发现限制和肿瘤基质定向重塑突发事件（视频1）。于肿瘤微环境开发力可能导致膨胀的肿瘤基质和后果重塑和肿瘤血管的类似程度的伸长或收缩，因为我们在愈合伤口11,24的情况下，先前显示。 视频1。扩展标记Ⅳ型胶原（红色）和腱糖蛋白C（青色）拉长血管（箭头）和被动转运三种肿瘤细胞（绿色）肿瘤基质 。的teascin C丰富的肿瘤基质易位血管（红色水平方向structur局部收缩e）在十二小时内成像约100微米。用免疫荧光显微镜采集图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/Video1_New.avi" target="_blank">点击此处查看视频。</a> 利用多光子显微镜与荧光标记的问题，因为在这些实验中使用的光子通量会很快漂白不受保护免受氧化25荧光染料。在这里，我们表明，我们可以执行双光子时间推移显微镜同时成像免疫标记腱糖蛋白C矩阵与荧光即使在高密度的光子双光子显微镜（视频2）的最小光漂白。 时双光子显微镜视频2。低电平的腱生蛋白Ç免疫荧光（红色）的光漂白。B16-F10-GFP的肿瘤细胞（青色）进行成像在打开背侧耳接种后9天。荧光信号通过应用抗坏血酸的保护林格氏缓冲到成像的组织。二次谐波产生（绿色）不重叠的带由生腱蛋白C。16位颜色深度的图像11中，4倍的平均z轴节用z-步骤1.9微米购入15分钟表示的新的矩阵。图像采集每1分钟5秒收集6 Z平面。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/Video2.avi" target="_blank">点击此处查看视频。</a> 我们还与成像转移性肿瘤细胞的免疫细胞间的相互作用。从荷瘤小鼠的肿瘤相关的血管渗出IV输液后8小时CMTPX标记的脾细胞，侵入肿瘤基质和积极互动与肿瘤细胞通过形成长效电池触点（视频2）。荧光基团-647标记的Ⅳ型胶原结构是耐在12小时成像的光漂白。 从瘤（B16-F10）-B CMTPX标记的脾视频3。互动抽穗用鼠标单个肿瘤细胞（GFP-B16-F10）。脾为四组织染色为IV型胶原输血后。 Ⅳ型胶原绿色（647），脾红（CMTPX），B16-F10青色（GFP）。用免疫荧光显微镜采集到的图像。录像时长12小时。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/Video3-corrected.mov" target="_blank">点击此处查看视频。</a> 新鲜分离的肿瘤细胞被覆盖在裸露耳真皮预先染色为IV型胶原。我们可以观察到，坚持组织后，一些群体的细胞开始沿血管和脂肪细胞的基底膜集体迁移。 视频4。肿瘤细胞沿基底膜迁移。正常耳皮肤染色胶原IV（647，红色）被覆盖有新鲜传代的B16-F10-GFP细胞和成象5小时。有些肿瘤细胞粘附于组织沿开始集体迁移血管及脂肪细胞的基底膜。用免疫荧光显微镜采集到的图像。视频时间:1.5小时<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51388/Video4.avi" target="_blank">点击此处查看视频。</a> 另外，由于耳真皮几乎是二维的，我们可以很容易地收集，而不是更慢和更昂贵的共聚焦或多光子显微镜大量使用快速荧光立体显微镜的数据。然而，它也可以使用任何上述扫描显微镜方法与我们的活体中频准备（ 图3，视频1）。

Watch this Scientific Journal Video about Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy at JoVE.com

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

细胞外基质在伤口愈合，炎症和肿瘤发生实质性的重塑。我们提出了一个新颖的活体免疫荧光显微镜方法来可视化纤维状的网状基质成分使用落射荧光或双光子显微镜的高空间和时间分辨率的动态以及。

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

long-term-intravital-immunofluorescence-imaging-tissue-matrix

Nanyang Technological University

Research

JoVE Journal

Bioengineering

19.0K 次观看.  École Polytechnique Fédérale de Lausanne. 细胞外基质在伤口愈合，炎症和肿瘤发生实质性的重塑。我们提出了一个新颖的活体免疫荧光显微镜方法来可视化纤维状的网状基质成分使用落射荧光或双光子显微镜的高空间和时间分辨率的动态以及。 

视频: Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

改善斑马鱼幼虫实验操作的长期活体成像装置

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

经济实惠的氧气显微镜辅助多细胞球体生物制造

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...