Name: fluorescence-quenching of a liposomal-encapsulated near-infrared fluorophore as a tool for in vivo optical imaging
Uploaded: 2015-01-05T15:00:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging at JoVE.com

这项工作是由德意志研究联合会授予 HI-698 / 10-1和RU-1652 / 1-1的支持。我们感谢琳五月优秀的技术援助和公司DYOMICS有限公司，耶拿的盛情支持。

注:所有的程序都是由区域动物委员会，并按照有关伦理使用动物的国际准则认可。 1.准备材料和仪器<ol><li>自发形成囊泡分散液的制备（SFV） <ol><li>溶解和制备原液以下磷脂:214毫克/毫升的蛋磷脂酰胆碱（EPC）134毫克/毫升胆固醇，122毫克/毫升1,2- distearoyl- SN -glycero -3- phosphoethanolamine- -N - [甲氧基（聚乙二醇）-2000]（铵盐）（MPEG 2000 -DSPE）和2毫克/毫升1,2-dioleoyl- SN -glycero -3- phosphoethanolamine- -N - （7-硝基2-1,3苯并恶-4-基）（铵盐）的氯仿，并存储在玻璃小瓶（NBD-DOP​​E）。 </li><li>提供大约3毫升氯仿在一个圆底烧瓶中并磷脂原液适当量转移到圆底烧瓶中，制备脂质体的EP组成C:澈:的mPEG 2000 -DSPE以6.5的摩尔比:3:0.5。脂质体双荧光标记添加0.3％（摩尔）NBD掺杂到脂质的解决方案。 </li><li>蒸发从减压（300毫巴）在55℃下使用旋转蒸发器将有机磷脂溶液中的氯仿。 </li><li>后，形成均匀的磷脂膜，降低压力至10毫巴1-2小时，以除去残留的氯仿存款。 </li><li>而氯仿蒸发，溶解DY-676-COOH（6.181微米）在10毫米的Tris缓冲液pH 7.4，并填写与液氮杜瓦瓶。接通超声波浴设定在50℃。 </li><li>传送DY-676-COOH（6.181微米）溶液到圆底烧瓶水合干燥的磷脂膜和涡大力直到自发形成囊泡（SFV）分散形式的适当体积（0.5-1毫升）中。确保所有的磷脂的分散，以避免脂质损失。 </li><li>仔细TRAnsfer圆底烧瓶含有SFV分散到液氮中并冷冻的分散3-5分​​钟。放置在圆底烧瓶中进在50℃的超声波浴中解冻该分散体然后旋涡振荡分散大力为1-2分钟。重复此步骤，六次，总共七个冻融循环。 </li></ol></li><li> SFV挤压，形成均匀的脂质体囊<ol><li>转移SFV分散到1ml注射器（注射器-a）和使用LiposoFast-基本挤出成注射器-b至100纳米的聚碳酸酯膜挤出的分散体。 </li><li>挤出从注射器-b中的分散性，回注射器一个，然后重复该循环10次。由于挤压，在从一个模糊的外观，以清晰的色散随时间的变化注射器的解决方案。经过十次（20单挤压步骤）从设备中取出注射器b和挤出分散从注射器直接上一次进入无菌1.5毫升反应管中。 </li></ol></li><li>脂质体的净化封装DY-676-COOH免费染料<ol><li>准备使用浸泡在10mM Tris缓冲液pH 7.4（柱长28厘米，直径0.8厘米）G25珠的柱色谱纯化。 </li><li>传送0.5毫升挤出囊泡分散体到凝胶床并且让样品漏极到凝胶基质中。 </li><li>洗脱用10mM Tris缓冲液pH为7.4（ 图1A）的脂质体和完全洗柱直至游离染料下水道出列。如果需要的话，收集并根据制造商的说明通过脱盐和脱水循环的游离染料。 </li><li>通过超速离心（200000×g离心2小时，在8℃）浓缩该洗脱的脂质体，然后它们分散在无菌的10mM Tris缓冲液的pH值，7.4足够体积。 </li></ol></li><li>封装DY-676-COOH浓度的定量<ol><li>通过溶解DY-676-COOH（0，82，124准备校准曲线，247，494，988纳米）在10mM Tris缓冲液，pH 7.4中含有0.1％的Triton X100。 </li><li>溶解的脂质体的2微升（的50毫摩尔/升的库存100纳摩尔最终脂质）中5分钟，在室温下，在含有1％Triton X100的破坏囊泡和释放包封的染料加入100μl的Tris缓冲液中。然后稀释用10mM Tris缓冲，pH为7.4至0.1％的最终的Triton-X100浓度样品（V / V）制备1毫升的总体积。准备好所有的样品一式两份。 </li><li>测量所有样品的吸收和发射（免费DY-676-COOH和Triton-X100处理的脂质体）在激发λ= 645 nm和发射λ= 700纳米。建立和使用游离染料的校准曲线来确定包封染料的浓度。 </li></ol></li><li>脂质体的表征<ol><li>确定脂质体通过动态光散射的大小和ζ电位。稀释的脂质体的样品与过滤灭菌（0.2微米）的10mM Tris缓冲液pH 7.4到交流oncentration 100-300微米（脂）。传送稀释的样品置于低体积一次性杯，并根据制造商的说明测量样品。 </li><li>通过电子显微镜表征脂质体根据标准方法来证明脂质体囊泡的大小，完整性和均匀性。 </li></ol></li></ol> 2.验证荧光淬灭，并准备脂质体的激活<ol><li>荧光猝灭和激活的理化分析<ol><li>准备两个1.5毫升管的唇-Q和2管免费DY-676-COOH。传输100纳摩尔总脂质（2.38微升的42毫摩尔/ L的唇-Q原液，含有包封的DY-676-COOH的138微克/毫升）成相应的管中。自由转会DY-676-COOH相当于唇-Q的染料含量（0.38微克导致例如，从138微克/ 1000微升2.38 X微升唇-Q使用）。孵育1桶每个探针在4℃下的e和冻结第二管在-80℃下过夜（16小时）。 </li><li>升温的加热块至30℃。填用碎冰冷却箱和平衡的10毫摩尔Tris缓冲液pH 7.4的等分试样至室温。 </li><li>从4℃除去探针和在室温下平衡（用铝箔包裹以避光），并迅速解冻探针-80℃，在30℃下进行5分钟。将它们转移至室温（也包在铝箔避光保护）之前寒意在冰上解冻的探针1分钟。 </li><li>添加的10mM Tris缓冲液（pH7.4）中，以各探针的100微升最终体积并平衡所有的探针在RT 10分钟。 </li><li>吸管80微升每种探针的进入低容积的玻璃比色杯中，并从上一个分光计400-900毫微米测量每个探测器的吸收。将探头返回到其相应的管中。 </li><li>转移80微升每个探针到合适的玻璃比色杯并测量通过激励探针上的荧光分光光度计的荧光发射在674 nm和从694-800纳米测量荧光。 </li></ol></li><li>细胞吸收和荧光激活<ol><li>根据标准条件（37℃，5％CO 2和95％潮湿气氛）得到并培养在其相应的培养基以下细胞系。这里，使用鼠巨噬细胞系J774A.1（Dulbecco改良的Eagle培养基补充有10％（V / V）胎牛血清），人成胶质细胞瘤细胞系，U-118MG（含有必需的维生素和10％MEM（体积/ ⅴ）胎牛血清）和人纤​​维肉瘤细胞系HT-1080（RPMI，用5％FCS）中。 </li><li>外套8孔腔室载玻片用聚L-赖氨酸（添加100微升0.001％聚-L-赖氨酸，以每孔，并孵育在37℃下进行10分钟。吸溶液并让室载玻片干涸至少4小时，在RT在无菌工作台，冲洗玻片3用200μlHank氏缓冲盐溶液次直到需要再密封它们与石蜡和铝箔，并储存在4℃。 </li><li>而该腔室载玻片枯竭，直到需要过滤消毒脂质体和染料溶液，并储存于4℃。 </li><li>探头摄取分析全身体内 NIR荧光成像系统，准备5个小培养瓶每个3测试细胞系（15瓶计）。种子2×10 6 J774A.1 U-118MG和每个培养瓶中的HT-1080细胞用5ml各自的培养基中（在五元组），并生长16-24小时。平行于培养瓶中分别接种30,000个细胞每个细胞系（J774A.1和HT-1080）或20,000个细胞（U-118MG）至2孔腔室载玻片中生长和在500μl培养基中16-24小时。 </li><li>第二天，加入唇-Q 100纳摩尔（脂质终量），以每细胞系，并在腔室载玻片每个细胞系的一个孔2烧瓶中。立即传输每个细胞系1烧瓶至4℃，并在第二个烧瓶回培养箱。 注:该探针的量添加到该烧瓶中，并在腔室玻片是相同的，使得上腔室玻片的浓度高10倍（5毫升是500微升培养基）。这是必要的，因为微观检测比在来自瓶中的细胞沉淀被成像的近红外荧光成像系统较不敏感。 </li><li>添加游离DY-676-COOH在相当于唇-Q的染料含量的细胞中每个细胞系，并在腔室载玻片每个细胞系的一个孔2烧瓶的浓度，然后立即传送每个细胞系1烧瓶至4℃（能量消耗），并一起另一烧瓶与腔滑动回到培养箱中。孵育所有细胞24小时，在相应的条件。将细胞在烧瓶中不含探针用作未处理的对照。 </li></ol></li><li> NIRF成像和半定量分析<ol><li>经过24小时培养的持续时间，通过用Hanks洗涤细胞2次收获细胞在烧瓶缓冲在500μl管盐溶液（HBSS），然后通过离心（5分钟，在200×g离心）刮去细胞在500μl的HBSS和沉淀。 </li><li>放置管子与细胞小球（和HBSS）在一个NIRF成像和图像使用滤波器对激励（615-665纳米）和发射（切&gt; 700纳米）。 </li><li>扣除自体荧光，并根据制造商的说明书评估靶的强度与自体荧光。这会给荧光强度作为平均信号（定标计数/秒），这代表缩放对于曝光时间，相机增益，分级和比特深度后计数的水平，从而使该测量是在彼此之间可比较的半定量水平。 </li></ol></li><li>共聚焦显微镜分析<ol><li>经过24小时培养后，洗涤2次，用500μL的HBSS收获上腔室载玻片中的细胞。 </li><li>修复了CELLS用含有3.7％（V / V）的甲醛进行30分钟，在室温加入200μlHBSS中。 </li><li>而固定是怎么回事，淡化色斑DNA，赫斯特-33258 1:50安装解决方案。 </li><li>固定后，洗涤细胞2次用HBSS，然后从玻璃载片分开的腔室。添加包含在对应于所述腔室玻片的孔中的每个点的DNA的染色50μl的安装方案。覆盖细胞用玻璃盖玻片，密封透明指甲油和风干10分钟边在室温（暗）。 </li><li>在合适的荧光显微镜或共焦显微镜图像的细胞。用于相应部件的可视化下面的激发和发射设置:原子核（赫斯特-33258:激发405纳米，发射420-480纳米）。 NBD-DOP​​E（脂质体脂质:激发488纳米，发射530纳米）。 DY-676-COOH（NIR荧光染料:激发波长633-645，650-700的发射波长）。 注:确保荧光显微镜vailable配备合适的过滤器，允许激发波长和发射超过630纳米高。 </li></ol></li></ol> 3.脂质体为基础的炎症活体荧光成像<ol><li>动物和材料的制备<ol><li>房子8-12周龄雄性NMRI小鼠体重与食物和水随意在标准条件下约36克</li><li>实验开始七天前，让所有小鼠的低脱镁叶绿饮食，以减少组织自发荧光。 </li><li>每个实验开始二十四小时之前，剃小鼠在期望区域（ 例如，整个如果后腿水肿成像所需的背部区域）。 </li><li>称重动物并计算探针的量将每只小鼠（LIP-Q和唇dQ的以10微摩尔（脂质浓度喷射）每公斤体重和自由DY-676-COOH（相当于染料的唇-Q使用的内容） 。 </li><li>图像中的动物全身NIR荧光成像器与用于细胞沉淀相同的设置。这种测量提供了动物的自发荧光。 </li><li>溶解10毫克酵母聚糖-A在1ml等渗盐水溶液和过夜储存于4℃。 </li></ol></li><li>诱导炎症和体内 NIRF成像<ol><li>准备每只小鼠3注射器包含以下解决方案。填充1注射器用50μl酵母聚糖A溶液（10毫克/毫升）和第二注射器用50μl等渗盐水溶液。与探针，即唇-Q和唇-DQ（10微摩尔/ kg体重（脂肪））被指定为试验动物和免费DY-676-COOH（集中在唇-Q）控制动物填补第三注射器。确保探针用无菌HBSS稀释至150微升最终体积。 </li><li>适用于动物的眼睛眼霜以避免干燥，麻醉动物用2％异氟醚，直到他们都深深睡着了，在爪子触摸时没有反应（这大约需要2分钟）。 </li><li>放置在鼠标上的暖垫（仍在麻醉）和注入的右后腿的酵母聚糖A溶液皮下和在左后腿的盐溶液。立即静脉注射探头和图像的动物之后再注入/测量记录时间（T = 0小时）。保存生成的图像作为图像数据集，并重复上述步骤，对所有其他动物和相应的探针。 </li><li>图像中的动物，每2小时，10小时后喷射，然后在24小时后喷射，确保在测量室的阶段是温暖（例如，通过将暖垫下方），为了避免低温。每次测量后，将动物的食物和水随意一个笼子里，放在笼中温带动物室。安乐死的动物通过首先麻醉用2％异氟烷，直到动物不再反应以触摸，然后用二氧化碳安乐死5-10分钟，使得确保动物停止呼吸完全尸僵发生。 </li><li>根据该可在线评估器官的标准协议（http://www.freebookez.com/mouse-dissection-lab-report/）和图像解剖小鼠。 </li><li>根据制造商的说明，通过首先扣除动物（去混合），然后分配的自体荧光的兴趣（左后腿与盐水）和发炎区域的目标荧光（右后腿与酵母聚糖-A）中的区域的整体荧光评估测量结果。 </li></ol></li></ol>

The authors have nothing to disclose.

因为脂质体还可以作为递送系统用于荧光染料，它们使目标疾病的成像。高浓度的荧光染料如NIRF染料，这里使用DY676-COOH的封装导致截留染料的荧光淬灭的高水平。荧光猝灭，这种现象以高浓度看到的许多荧光团可被利用在几种体内成像应用，其中，具有高灵敏度的目标区域的可靠的检测被征求。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。 ...

脂质体已被广泛研究，并作为最生物相容性生物医学的药物递送系统的临床应用1,2之一。它们主要由磷脂和胆固醇，这两者都是生物相容的化合物模仿天然细胞膜份。而亲水性物质可在含水内部被截留，亲脂性试剂可以脂质体磷脂双层3内被引入。脂质体的含水内部中的封装的物质给予保护，防止降解在体内 ，也防止了主机系统从用于疾病的治疗的细胞毒性药物，例如化学治疗旨在破坏肿瘤细胞的毒性作用。脂质体表面与像聚乙二醇聚合物的修饰（PEG化）进一步延伸体内的脂质体的血液循环时间，由于空间位稳定4。 Moreov呃脂质体可隔离的高浓度的几种物质，如蛋白质5,6，亲水性物质7,8和酶9。因此，它们作为可靠的临床治疗和诊断工具，值得他们的批准用于递送细胞毒性药物，如阿霉素治疗癌症4。由于其灵活性，脂质体还可以装载有荧光染料用于诊断和图像引导手术目的。 荧光成像提供了一个高性价比的和非侵入体内诊断工具，但需要一些基本要求。它可以证明，这适合最适合于体内成像的荧光染料具有特征吸收和发射最大值在光分散和散射，以及组织自发荧光从水始发和血红蛋白是低的范围内。因此，这样的探针具有其650和900纳米之间10 ABS /青霉最大值。<html>除此之外，无论是在体外和体内的荧光染料的稳定性是至关重要的，如调理作用和快速清除可大大限制了它们用于体内成像应用11。其他效果，例如差的稳定性和低灵敏度或观察与吲哚花青绿（ICG）12-16对靶器官的细胞毒作用，是不希望的，并用于体内成像设计探针时，必须考虑到。这些观察导致了若干临床前的近红外荧光染料，纳米颗粒以及用于体内成像的炎性过程，癌症和用于图像引导手术17-20新技术的积极发展。尽管大多数的临床前NIRF（近红外荧光）的稳定性的染料在体外 ，其通过肝脏和肾脏快速灌注和清除阻碍它们在疾病和炎症过程的体内光学成像的使用。 ntent"&gt;因此，我们提出了一个协议对荧光染料的封装，如良好表征的近红外荧光染料DY-676-COOH，在脂质体以其倾向自淬火，在相对 ​​高浓度的21，在高浓度的H-二聚体形成和/或PI-层叠荧光团分子的增加之间位于彼此的福斯特半径结果在荧光分子之间荧光共振能量转移（FRET）内的荧光团的分子之间的相互作用，在低浓度的空间中，从而防止PI-堆积作用和H-二聚体形成并导致高的荧光发射。高，低浓度和伴随荧光猝灭和激活之间的开关是一种很有前途的策略，可被利用为光学成像22上 。在这方面，封装的高浓度的NIRF染料的DY-676-COOH在脂质体的含水内部更发vorable用于体内成像比游离染料。该方法的挑战在于在正确的封装首先和其次，在从包封高浓度的染料产生的利益的验证。与游离染料与非淬火脂质体制剂与低浓度的染料的比较猝灭脂质体的成像特性，也是必不可少的。我们显示通过简单的，但高度有效膜水化和挤压协议结合交替冻结和解冻循环是DY-676-COOH的淬火浓度的脂质体中的封装是可行的。用于制备脂质体的其它方法，如逆相蒸发法23以及乙醇注入法24使脂质体制剂具有高包封率的许多亲水性的物质。然而，要被封装的物质的性质可以影响所述包封效率。实际上，这里介绍的薄膜水化和挤压协议揭示了DY-676-COOH的封装效率最高。为了说明DY-676-COOH，一个酵母多糖诱发的水肿模型，它允许在几个小时内的炎症过程的研究的脂质体包封的好处，被使用。这里，它被证明脂质体与高浓度的包封的DY-676-COOH是在炎症过程中比游离染料或未淬火脂质体制剂具有低染料浓度的体内光学成像更适合于全身。因而底层协议提供了一种简单而快速的方法，以产生荧光猝灭脂质体和在体外和体内的活化和成像潜在的有效性。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials and equipments for preparation of liposomes&lt;/strong&gt;</td></tr><tr><td>egg phospahtidylcholine</td><td>Avanti Polar Lipids</td><td>840051P</td><td>Dissolve in chloroform and store in glass vials (214 mg/ml)</td></tr><tr><td>cholesterol</td><td>Sigma</td><td>C8667</td><td>Dissolve in chloroform and store in glass vials (134 mg/ml)</td></tr><tr><td>1,2-distearoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-[methoxy(polyethylene glycol)-2000] (ammonium salt)</td><td>Avanti Polar Lipids</td><td>880120P</td><td>Dissolve in chloroform and store in glass vials (122 mg/ml)</td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)</td><td>Avanti Polar Lipids</td><td>810145P</td><td>Dissolve in chloroform and store in glass vials (2 mg/ml)</td></tr><tr><td>Sartorius MC1 (d = 0.01 mg)</td><td>Sartorius AG</td><td>Research RC 210 P</td><td>used for weighing the phospholipids</td></tr><tr><td>Rotavapor</td><td>B&amp;uuml;chi Labortechnik AG</td><td>R-114</td><td>used for hydration of phospholipid film</td></tr><tr><td>Waterbath</td><td>B&amp;uuml;chi Labortechnik AG</td><td>R-481</td><td>used for hydration of phospholipid film</td></tr><tr><td>Vacuum Controller</td><td>B&amp;uuml;chi Labortechnik AG</td><td>B-720</td><td>used for hydration of phospholipid film</td></tr><tr><td>Vacobox</td><td>B&amp;uuml;chi Labortechnik AG</td><td>B-177</td><td>used for hydration of phospholipid film</td></tr><tr><td>Circulation Chiller</td><td>LAUDA DR. R. WOBSER&lt;br/&gt; GMBH &amp;amp; CO. KG</td><td>WKL 230</td><td>used for hydration of phospholipid film</td></tr><tr><td>DY-676-COOH</td><td>Dyomics GmbH</td><td>676-00</td><td>Dissolve in 10 mM Tris and store stock at -20&amp;deg;C</td></tr><tr><td>Tris-(Hydroxymethyl)-aminomethan</td><td>Applichem</td><td>A1086</td><td>buffer 10 mM, pH 7.4</td></tr><tr><td>Trichlormethan</td><td>Carl Roth GmbH + Co. KG</td><td>Y015.2</td><td>used for liposome preparation</td></tr><tr><td>Sonicator</td><td>Merck Eurolab GmbH</td><td>USR 170 H</td><td>used for liposome preparation</td></tr><tr><td>Vortex Genie 2 (Pop-off Cup, No. 146-3011-00)</td><td>Scientific Industries Inc.</td><td>SI-0256</td><td>used for liposome preparation</td></tr><tr><td>Sephadex G25 medium&amp;nbsp;</td><td>GE Healthcare Europe GmbH</td><td>17-0033-01</td><td>used for liposome purification</td></tr><tr><td>Triton X100</td><td>Ferak Berlin GmbH</td><td>505002</td><td>used to destruct liposomes&amp;nbsp; for dye quantification</td></tr><tr><td>LiposoFast-Basic</td><td>Avestin Inc.</td><td></td><td>used for the extrusion of liposomes</td></tr><tr><td>Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U)</td><td>VWR</td><td></td><td>used for the extrusion of liposomes via LiposoFast-Basic</td></tr><tr><td>Fluostar Optima</td><td>BMG Labtech</td><td></td><td>used for dye quantification</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td></td><td>used for the determination of liposome size and zetapotential</td></tr><tr><td>Ultracentrifuge&amp;nbsp;</td><td>Beckmann Coulter GmbH</td><td>XL 80</td><td>used for concentration of the samples</td></tr><tr><td>Rotor</td><td>Beckmann Coulter GmbH</td><td>SW 55 TI</td><td>used for concentration of the samples</td></tr><tr><td colspan="4">&lt;strong&gt;Materials and equipments for the evaluation of liposome and optical imaging&lt;/strong&gt;</td></tr><tr><td>Zymosan-A from Saccharomyces cereviciae</td><td>Sigma</td><td>Z4250-250MG</td><td>used for induction of inflammation</td></tr><tr><td>Isotonic Saline (0.9%)</td><td>Fresenius GmbH</td><td>PZN-2159621</td><td>used for the dilution of Zymosan-A</td></tr><tr><td>Isoflurane vaporizer</td><td>Ohmeda Isotec 4</td><td></td><td>used for anesthesizing animals</td></tr><tr><td>Isoflurane</td><td>Actavis GmbH&amp;nbsp;</td><td>PZN-7253744</td><td>anesthesia</td></tr><tr><td>Thermo Mat Pro 20 W</td><td>Lucky Reptile</td><td>61202-HTP-20</td><td>used to keep animals warm during anesthesia</td></tr><tr><td>Omnican-F (1 ml injection)&amp;nbsp;</td><td>Braun</td><td>PZN-3115465</td><td>used for subcutaneous and intravenous application of probes</td></tr><tr><td>Panthenol eye cream</td><td>Jenapharm</td><td>PZN-3524531</td><td>used to prevent dryness of the eyes of animals during anesthesia</td></tr><tr><td>Hanks buffered saline solution</td><td>PAA Laboratories /Biochrom AG</td><td>L2045</td><td>w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells</td></tr><tr><td>8-Well chamber slides</td><td>BD Biosciences</td><td>354108</td><td>used for cell culture followed by microscopy&amp;nbsp;</td></tr><tr><td>Cell culture flasks</td><td>Greiner BioOne</td><td></td><td></td></tr><tr><td>Cell culture media</td><td>Gibco (life technologies GmbH)</td><td></td><td></td></tr><tr><td>Fetal calf serum&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Poly-L-Lysine solution (0.01%, 50&amp;nbsp;ml)</td><td>Sigma</td><td>P4832</td><td>used to coat cell culture chamber slides</td></tr><tr><td>Mountant Permafluor</td><td>ThermoScientific&amp;nbsp;</td><td>S21022-3</td><td>Mounting solution for microscopy</td></tr><tr><td>Hoechst-33258</td><td>AppliChem</td><td></td><td>DNA stain for microscopy</td></tr><tr><td>Hera-Safe</td><td>Heraeus Instruments</td><td></td><td>sterile work bench used for cell culture</td></tr><tr><td>HERA cell</td><td>Heraeus Instruments</td><td></td><td>Incubator used for cell culture</td></tr><tr><td>LSM510-Meta</td><td>Zeiss</td><td></td><td>used for confocal microscopy</td></tr><tr><td>Maestro-TM in vivo fluorescence imaging system</td><td>CRi, Woburn</td><td></td><td>used for whole body fluorescence imaging of small animals</td></tr><tr><td>Spectrophotometer (Ultrospec 4300 pro UV)</td><td>GE Healthcare</td><td></td><td>used for measurement of absorption</td></tr><tr><td>Spectrofluorometer (Jasco FP-6200)</td><td>Jasco</td><td></td><td>used for measurement of fluorescence emission</td></tr><tr><td colspan="4">&lt;strong&gt;Animals&lt;/strong&gt;</td></tr><tr><td>NMRI mice (8-12 weeks old, male)</td><td>Elevage Janvier, France</td><td></td><td>used for inflammation trials</td></tr></tbody></table>

fluorescence-quenching of a liposomal-encapsulated near-infrared fluorophore as a tool for in vivo optical imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

高浓度的荧光染料，如这里所使用的脂质体的含水内部的NIRF染料DY676-COOH的封装导致荧光猝灭的一个高的水平。荧光猝灭，这种现象可见有许多荧光团以高浓度，可以利用几种体内成像应用，其中一个高灵敏度的目标区域的可靠的检测要求较高。使用脂质体还提供了保护，这是不可缺少的用于体内应用的染料。脂质体的彻底的表征是必要的，包括几个因素，如染料包封，稳定性和脂?...

Watch this Scientific Journal Video about Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging at JoVE.com

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

fluorescence-quenching-liposomal-encapsulated-near-infrared

Research

JoVE Journal

Bioengineering

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

16.2K 次观看.  Jena University Hospital. The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity. 

视频: Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

科研

免疫与感染

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

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

荧光显微镜对聚焦超声的刺激

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

工程学

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

医学

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

<meta charset="utf8"></head><body>用于 smFRET-TIRF 研究的 RNA 双色末端标记和囊泡封装

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

<meta charset="utf8"></head><body>用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

生物化学

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

摘要

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此视频中的章节

非侵入性光学成像淋巴血管的鼠标

一种超越BRET步骤：通过荧光激发无限制自发光（FUEL）

荧光显微镜对聚焦超声的刺激

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

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用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

利用pHluorin标记的受体监视亚细胞定位和贩运

荧光淬一个脂质体包封的近红外荧光团的作为一个工具

摘要

探索更多视频

此视频中的章节

非侵入性光学成像淋巴血管的鼠标

一种超越BRET步骤：通过荧光激发无限制自发光（FUEL）

荧光显微镜对聚焦超声的刺激

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

无创

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

用于单分子 FRET-TIRF 显微镜的长 RNA 的荧光末端标记和封装

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

利用pHluorin标记的受体监视亚细胞定位和贩运

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像