The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

伦理学声明:以上所描述的，根据经批准的协议，并在纪念斯隆-凯特琳癌症中心（MSK）机构动物护理和使用委员会（IACUC）的道德准则进行体内动物实验。 1.一雾化装置的构建<ol><li>木连接部分A（12.5×2.5×1.8 cm 3）的直立用两个螺丝（4×25 平方毫米）B部分（12.7×10.7×1.8 立方厘米 ）的中心。附上木质部-C（11×2.5×1.8 毫升 ），以利用一个螺钉，使得C部分（11×2.5×1.8 毫升 ）仍可被移动部A（12.5×2.5×1.8 毫升 ）的中间。 见图1。 </li><li>钻两个孔通过塑料3盎司微型喷雾器（D）的喷射瓶扳机的下部末端，并按下一个不锈钢棒（10厘米的1/16"钢）（E）通过，以形成两个环，一个在任一触发器的一侧。 </l我&gt; <li>包住喷射瓶用胶带（F）的底部，以防止滑出扎带。附加喷雾瓶使用两个塑料扎带木质部C（11×2.5×1.8 毫升 ）（28厘米）（G）。 </li><li>切断011伺服电机（Ⅰ）中，用伺服控制（H）的松散的电缆重新连接。然后，伺服电机连接至用胶带木质部A（12.5×2.5×1.8 毫升 ）的顶部。 </li><li>附加铅笔（J）使用回形针（K）伺服电机控制杆。紧密连接的铅笔用塑料盖扭线（L）钢棒循环的最外层部分和安全的用胶带铅笔的末端。 </li><li>切断伺服电机控制单元的磁电缆连接器（M），并将其重新连接到扬声器连接线（N）。然后，伺服电机控制部胶带到木质部B（12.7×10.7×1.8 毫升 ）。切断与磁性连接器的W1的电缆在一半和附加一个部分在铜S的松散端调峰电缆（1米）。连接（磁）12拨动开关和P1的电源可用W1电缆和一个9V电池。 </li></ol> 2.方法的灵敏度测定<ol><li>在1.5毫升离心管中，在260微克（347纳摩尔），52微克（69纳摩尔），26微克（34纳摩尔）金额反渗透水（100微升）准备[茹（联吡啶）3] Cl 2的的解决方案，5微克（6.9纳摩尔），3微克（3.5纳摩尔），520纳克（694皮摩尔），260纳克（347皮摩尔），52纳克（69皮摩尔），26纳克（34皮摩尔），5毫微克（6.9皮摩尔）和3纳克（3.5皮摩尔）。 </li><li>混合100μL的每个的[Ru（联吡啶）3] Cl 2中的溶液与100微升硝酸铵铈的水溶液（（NH 4）2的Ce（NO 3）6）在显微镜载玻片上的水（25毫摩尔）。 </li><li> 通过初始化成像软件建立在生物发光读者收购。 <ol><li>登录到用户配置文件，并寻找阿奎习得的控制面板。点击"初始化"，等到仪器准备。 </li><li>寻找"拍摄模式"，并确保"发光"和"照片"被选中，而"荧光"是没有选。 </li><li>改变"曝光时间"为"发光"设置为20秒。设置"发光"其余的设置如下:"分档":中等; "F /停止":1;和"排放过滤器":打开。 注:曝光时间可能需要根据仪器和所用的实验设置，可以适于，如果从呈现的设置不同。 </li><li>对于"照片"，请使用以下设置:"接触时间":自动; "分档":中等;和"F /停止":8.对于"视场"下拉菜单成像target.Look调整"主题高度"。初始设置为"C"。 C在更改为"B"（14厘米的距离相机和样品台）。 </li></ol></li><li> 通过将显微镜载玻片上黑施工纸张上成像室以保护其免受氧化剂的地板的片材设置的喷雾器。混合100μLdroplet的[茹（联吡啶）3] Cl 2的-solution用100微升的水溶液（NH 4）2铈（NO 3）6.观察绿框十字线。 <ol><li>放置在黑色施工纸成像对象，使得所感兴趣的区域是在绿色光盒瞄准器的中心显示在样品台上。由木支持卸下塑料喷雾瓶准备喷雾器。补的三乙胺中的溶液（1:3的水/乙醇）成其塑料容器和其重新安装到木支撑。 </li><li>将生物发光读取器内部雾化器，确保电源线从电源线雾化器断开。确保电源开关是，拨动开关处于关闭状态，红色LED亮起。放置喷雾器，使得喷射流指向朝向上的成像对象感兴趣的区域，同时尽量减少从摄像机朝向由喷嘴头的成像对象的视图阻塞。 </li><li>将在任何潜在的热点黑色小片建筑用纸（如白色的痕迹在显微镜载玻片或注射部位），从喷雾保护他们。地点的至少40厘米的成像室内部的喷雾器遥控线的，使得它不与成像对象，喷雾器，或磁性门闩干扰。关闭成像系统的门。 注意:十字线将根据"视场"改变大小设置"生活图片;"确保此设置为"B"。 </li></ol></li><li>通过启动成像序列获取图像。点击"获取的"中"采集控制面板"。在第一摄像色曲ENCE，如果需要的话（推荐）使自动保存，并选择一个数据文件夹。忽略"编辑影像标签"对话直到序列的结束。 注:控制软件显示仪表的行动一步一步的实时性。制备测量和移动所述样本台，以合适的位置之后，它打开相机快门和计数测量时间。活门开启也可以通过由机器产生的咔嗒声听到。 </li><li>通过切换切换开关三次，以产生化学发光:在快门打开时，喷三乙胺的溶液的三连发（在水/乙醇3，每喷突发0.24±0.04毫升1）。 注:样品阶段将在测量过程中移动。留出足够的（最少40厘米）的电缆在仪器内部，让这一点。确保该溶液通过喷雾器喷洒可由上升管被渴望并有在管道中没有气泡。有几个备用BATTERIES为喷雾器准备需要的情况下。 </li></ol> 3. 在体内成像全身静脉注射后<ol><li>在1.5毫升微量离心管中，制备100微升含有的[Ru（联吡啶）3] Cl 2中的8和33之间纳摩尔磷酸盐缓冲盐水（PBS）溶液。制备的水溶液（NH 4）2的Ce（NO 3）中的同时用水（25毫摩尔）6。 </li><li>静脉内注射100微升的[Ru（联吡啶）3] Cl 2中成健康小鼠的尾静脉中（N = 5）。 </li><li> 通过CO 2窒息注射后安乐死的小鼠10分钟。 <ol><li>从躯干Y切割取下皮肤，然后取出肋弓的U形，露出心脏和肺部。通过一个24号针头在右心房切割的出口和注入20毫升的PBS到左心室18灌注小鼠。通过腹部经过精心切割皮肤和揭露肾脏和肝脏。纵向切开通过机关创建一个可见的削减。 </li></ol></li><li> 如步骤2.3-2.6中所述，做出了以下改变设置的采集。 <ol><li>彻底冲洗喷雾器塑料储之后，具有的（NH 4）2的Ce（NO 3）6在水代替三乙胺溶液（25毫摩尔）填充。 注意:要彻底冲洗每次使用后喷嘴雾化器，因为结晶是很重要的（NH 4）2铈（NO 3）6可经过多次使用破坏喷嘴。 </li></ol></li><li> 使用整个动物或器官样品进行成像。 <ol><li>对于全腹成像，定位与开腹面对相机，指着仪器的后脑勺鼠标尸体。居中机关在绿色灯箱十字线成像（ 例如，肝脏或肾脏）。 </li><li> F或单个器官成像和量化，从成像仪器中取出鼠标和针下来。从已经开体腔开始，切除内部器官（ 例如，肾，肝，肺，肌肉，脾脏，脑，和心脏）。通过后腿皮肤切开切除的肌肉组织。小心打开颅骨用手术刀切除大脑。 <ol><li>如果感兴趣的器官是肝脏，肾脏，脾，切成两半所有器官纵向，将每个器官的培养皿或一块黑色的建设纸。 </li></ol></li><li>按照步骤2.3-2.6建立单个器官的化学发光示踪剂的相对发射描述的过程。 </li></ol></li></ol> 4.淋巴结体内成像<ol><li>制备含有80纳摩尔的[Ru（联吡啶）3] Cl 2中的一个的PBS溶液10μL。制备的水溶液（NH 4）2的Ce（NO 3）6在华之三（25毫米）在同一时间。 </li><li>注入溶液10μL皮下到健康小鼠的后爪（N = 5）。作为阴性对照，注射用纯净的PBS10μL的对侧爪。注射后通过二氧化碳窒息牺牲小鼠15分钟。在两条后腿去除皮肤暴露淋巴管至腘窝淋巴结。 </li><li>如步骤3.4所述设置了收购。 </li><li>从两个后腿除去腘淋巴结，切断他们在一半，并与氧化剂喷他们上的培养皿，如之前（步骤3.5.3）中描述的，用于定量的目的。 </li></ol>

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Organomet. Chem. 744, 82-91 (2013).</li><li>Aoki, Y., 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=An+experimental+xenograft+mouse+model+of+diffuse+pontine+glioma+designed+for+therapeutic+testing.">An experimental xenograft mouse model of diffuse pontine glioma designed for therapeutic testing.</a> J Neurooncol. 108 (1), 29-35 (2012).</li><li>Forster, R. J., Bertoncello, P., Keyes, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrogenerated+Chemiluminescence.">Electrogenerated Chemiluminescence.</a> Annual Rev. Anal. Chem. 2, 359-385 (2009).</li><li>Connell, T. U., James, J. L., White, A. R., Donnelly, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Labelling+with+Versatile+Phosphorescent+Metal+Complexes+for+Live+Cell+Luminescence+Imaging.">Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.</a> Chem. Eur. J. 21 (40), 14146-14155 (2015).</li><li>Zhou, X., 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=Synthesis,+labeling+and+bioanalytical+applications+of+a+tris(2,2&#8242;-bipyridyl)ruthenium(II)-based+electrochemiluminescence+probe.">Synthesis, labeling and bioanalytical applications of a tris(2,2&#8242;-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.</a> Nat. Protoc. 9 (5), 1146-1159 (2014).</li><li>B&#339;uf, 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=Encapsulated+Ruthenium(II)+Complexes+in+Biocompatible+Poly(d,l-lactide-co-glycolide)+Nanoparticles+for+Application+in+Photodynamic+Therapy.">Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.</a> ChemPlusChem. 79 (1), 171-180 (2014).</li><li>Loizidou, M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+its+applications+in+surgery.">Nanotechnology and its applications in surgery.</a> Brit. J. Surgery. 97 (4), 463-465 (2010).</li><li>Barry, N. P. E., Sadler, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+for+Metals+in+Medicine:+How+Nanotechnology+May+Help+To+Shape+the+Future.">Challenges for Metals in Medicine: How Nanotechnology May Help To Shape the Future.</a> ACS Nano. 7, 5654-5659 (2013).</li><li>Hasan, K., Bansal, A. K., Samuel, I. D. W., Rold&#225;n-Carmona, C., Bolink, H. J., Zysman-Colman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Emission+of+Cationic+Iridium+(III)+Complexes+Towards+the+Red+Through+Methoxy+Substitution+of+the+Cyclometalating+Ligand.">Tuning the Emission of Cationic Iridium (III) Complexes Towards the Red Through Methoxy Substitution of the Cyclometalating Ligand.</a> Nat.Sci. Rep. 5, 1-15 (2015).</li><li>Truong, 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=Chemiluminescence+detection+with+water-soluble+iridium(III)+complexes+containing+a+sulfonate-functionalised+ancillary+ligand.">Chemiluminescence detection with water-soluble iridium(III) complexes containing a sulfonate-functionalised ancillary ligand.</a> Analyst. 139 (22), 6028-6035 (2014).</li></ol>

The authors have nothing to disclose.

这里，我们已经提出了一种技术，能够通过由化学发光记者创建的光子的发射光学划定组织。相对于其他，更成熟的，技术4，5，6，7，8，9，此化学发光报道系统采用了成像探针是无放射性和在非常高的灵敏度水平便于检测。或许更重要的是，化学...

在近几十年中，成像技术已经彻底改变，医生诊断和监测疾病的方法。这些成像技术，然而，已经在很大程度上仅限于全身成像系统，例如正电子发射断层扫描（PET），单光子发射计算机断层摄影（SPECT），计算机断层扫描（CT）和磁共振成像（MRI）。特别注意了癌症，而技术突破成像有很大的提高，这种病诊断和治疗的方式。尽管有这些进展，但是一个地方，这些成像技术只是不适合:手术室。虽然全身成像技术可在手术计划帮助下，他们通常缺乏空间分辨率足够高，以帮助医生实时确定是否所有的肿瘤组织已被删除或残留的肿瘤组织仍然在手术切缘1隐藏。确保无浸润肿瘤边缘被留下是最重要的手术目标之一，和外科医生必须走严格而谨慎的组织切除之间的紧绳子。如果太多被去除，不需要的副作用对患者正在加剧;如果太少被删除，复发率增加2，3。因此，划定精确的肿瘤边缘是至关重要的，而且我们相信，化学发光成像术可通过帮助医生形象化恶性组织，否则会没有被发现已建立的技术，以提高肿瘤边缘的识别精度。 目前正在调查其可能的实用程序，术中成像系统的许多影像技术。这些包括β-和γ射线发射探头4，光学荧光5，拉曼光谱6 </sup&gt;，7和切伦科夫发光8,9。迄今为止，然而，没有这些都成为确立为标准的临床工具。光学荧光成像迄今被证明是最有前途的这些技术，因此是最探讨。虽然它已经被证明是对许多应用的有价值的工具，它也不是没有局限性。事实上，它的主要缺点是由固有的自发荧光的生物组织所产生的背景荧光。这样的背景自发荧光信号是周围组织的激励的产物，除了荧光团，通过所需要的荧光信号的产生的外部光源。从实践的角度来看，这自发荧光可以潜在地导致低信号对噪声比，它可以限制在手术室这种技术的效用。 校长优势化学发光成像在荧光成像是没有激发光是必要的。其结果是，没有背景自发荧光。在化学发光成像，代替化学生成的激发能量。这一过程不会产生意外的背景信号，因此可以导致更高的信号 - 噪声比。这可能最终导致手术切缘的更精确和准确的检测。出人意料的是，这种方法作为一种手术成像技术的效用仍然未开发的10。事实上，最近的例子，这种技术是鲁米诺由髓过氧化物酶的小鼠11，12，13的氧化。因此化学发光生物医学成像是研究的一个，而未开发地区，可以具有以下优点:（1）导致与喜低背景信号最小自发荧光gher信号 - 噪声比; （2）化学发光发射范围从可见光到近红外的波长可调波长; （3）官能化的化学发光复合物，当与连接器的技术相结合，有针对性已经存在的生物分子，靶向分子成像探针14的整个库提供接入。 验证的原理该研究说明了化学发光成像在使用基于钌 - 成像剂的生物医学设置的潜在效用。该化合物的化学发光特性很好的研究，以调查可追溯至60年代中期15。经化学活化，所述试剂产生在约600毫微米16的光，这是非常适合用于医疗成像目的。活化能是由导致激发态-其具有650纳秒寿命在水中17 -foll的氧化还原反应提供在此激发态的松弛由光子的产生欠款。通过使用一个特别设计的远程喷雾器的，我们能够检测到化合物既体外 和体内 。最初的实验的结果是非常有前景的，这表明该技术的进一步调查。

<table><tbody><tr><td>Wood part A (12.5 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>&amp;nbsp;Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part B (12.7 &amp;times; 10.7 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part C (11 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Screws (4 &amp;times; 25 mm)</td><td>Screwfix</td><td>79939</td><td></td></tr><tr><td>Harmon Face Values 3 oz mini sprayer</td><td>Bed, Bath and Beyond</td><td></td><td></td></tr><tr><td>stainless steel rod (10 cm of 1/16&amp;rdquo; steel)</td><td>Metals Depot Int. Inc.</td><td>2192</td><td></td></tr><tr><td>Pencil Classic HB</td><td>Papermate</td><td>58592</td><td></td></tr><tr><td>Paper clip</td><td>Office Depot</td><td>221720</td><td></td></tr><tr><td>speaker cable</td><td>RCA Inc.</td><td>AH1650SN</td><td></td></tr><tr><td>Energizer 9V alkaline battery</td><td>Energizer Holdings Inc.</td><td>EN22</td><td></td></tr><tr><td>Hitech HS-82MG Micro Servo Motor, 3.4 kg/cm output torque @ 6V</td><td>Hitech RCD USA Inc.</td><td>32082S</td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalog Number</td><td>Comments</td></tr><tr><td>28 cm plastic cable ties</td><td>General Electric Inc.</td><td>50725</td><td></td></tr><tr><td>Duct tape</td><td>3M Inc.</td><td>3939</td><td></td></tr><tr><td>littleBits w1 wire</td><td>littleBits Inc.</td><td>w1 wire</td><td></td></tr><tr><td>littleBits p1 power</td><td>littleBits Inc.</td><td>p1 power</td><td></td></tr><tr><td>littleBits i2 toggle switch</td><td>littleBits Inc.</td><td>i2 toggle switch</td><td></td></tr><tr><td>littleBits 011 servo</td><td>littleBits Inc.</td><td>011 servo</td><td></td></tr><tr><td>20 cm plastic covered wire twist ties</td><td>Four Star Plastics</td><td>71TIE8000</td><td></td></tr><tr><td>Tris(2,2&amp;prime;-bipyridyl)dichlororuthenium(II) hexahydrate</td><td>Sigma-Aldrich Inc.</td><td>224758</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Ammonium cerium(IV) nitrate</td><td>Sigma-Aldrich Inc.</td><td>22249</td><td></td></tr><tr><td>Isofluorane</td><td>Baxter Healthcare</td><td>1001936060</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>PBS1</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich Inc.</td><td>T0886</td><td></td></tr><tr><td>Water</td><td></td><td></td><td>Water was purified using a Milipore Mili-Q (R &amp;ge; 18 M&amp;Omega;)</td></tr><tr><td>Female nude (outbred) mice</td><td>Jackson Laboratories</td><td>1929</td><td>age 5 - 6 weeks</td></tr><tr><td>Strain C57BL/6J&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>NU/J male mice at&amp;nbsp;</td><td>Jackson Laboratories</td><td>2019</td><td>age 6 &amp;ndash; 8 weeks</td></tr><tr><td>IVIS 200 bioluminescence reader</td><td>Caliper Live Science</td><td></td><td></td></tr><tr><td>Live Image 4.2 software</td><td>Perkin-Elmer</td><td>128165</td><td></td></tr><tr><td>Microscope slides</td><td>ThermoScientific</td><td>4951PLUS4</td><td></td></tr></tbody></table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

在协议部分1中所述的喷雾器系统可以从易得的材料以低成本来构造。它旨在是用于远程触发的生物发光读卡器内的还原/氧化剂的喷射（ 图1）的插入。我们的设计允许生物发光读者内的喷雾器在从透镜14厘米的距离的安全运行。在操作过程中，观察到透镜的不起雾或模糊。我们选择了市售化学发光剂的[Ru（联吡啶）3] Cl 2中为我们的方法是根据...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

a-novel-technique-for-generating-observing-chemiluminescence

Research

JoVE Journal

Bioengineering

8.4K 次观看.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

视频: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遗传学

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

医学

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

就地生物发光细菌细胞密度与光发射的测量与相关性研究

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生物化学

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

一种超越BRET步骤:通过荧光激发无限制自发光（FUEL）

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

生物工程

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

在生物环境的新技术的产生和观测化学发光

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