The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

伦理声明:以上所描述的体内动物实验是根据经批准的协议，并在纪念Sloan Kettering癌症中心的机构动物护理和使用委员会（IACUC）的道德准则进行。 1.合成TZ-BN-NOTA的<ol><li>在一个小的反应容器中，在600微升的NaHCO 3缓冲液（0.1M，pH值8.1）溶解7毫克的NH 2 -Bn-NOTA（1.25×10 -2毫摩尔）。检查溶液的pH值。如果需要的话，用0.1M的钠2小等份CO 3调节溶液的pH至8.1。 </li><li>在NH 2 -Bn-NOTA解决方案添加0.5毫克TZ-NHS（1.25×10 -3毫摩尔）在1.7 ml离心管。 注:TZ-NHS既可以称出干燥或无水的DMF或DMSO（&lt;50微升）的储备溶液。 </li><li>允许将得到的反应溶液中进行30分钟，在RT下的反应适度搅拌。 </li><li> 30分钟后，使用纯化逆相C 18 HPLC色谱以除去未反应的NH 2 -Bn-NOTA的产物。所述NH 2 -Bn-NOTA可以在254纳米的波长进行监控，而TZ-NHS和TZ-BN-NOTA最好在525纳米的波长监测。 注:保留时间明显高度依赖于每个实验室（泵，柱，管， 等等 ）的高效液相色谱法的设备的设置。然而，到目前的例子中，如果为0的梯度:100的MeCN / H 2 O（均含有0.1％TFA）至100:0的MeCN / H 2 O在25分钟内和分析4.6×250毫米C18 柱使用，TZ-BN-NOTA，TZ-NHS，和NH 2的保留时间-Bn-NOTA将大约15分钟，16.5分钟和10分钟，分别。该产品可以与其它反应组分中任一单次运行或使用半制备或制备-C 18 HPLC柱多个运行进行纯化。1 H-NMR，肛门ytical HPLC和ESI-MS是可用于验证完成TZ-BN-NOTA前体的纯度的所有方法。11 </li><li>冻结使用液氮收集HPLC洗脱液。 </li><li>包装在不透明的铝箔冷冻收集管。 </li><li>放置在冷冻干燥容器澳冰冻收集管/ N去除HPLC流动相。 </li><li>存储所述纯化的产物（一个明亮的粉红色固体）在黑暗中在-80℃。 注意:这是在程序中可接受的停止点。已完成的TZ-BN-NOTA的前体可稳定至少1年在这些条件下。 </li></ol> 2.准备huA33-TCO免疫偶联物<ol><li>在1.7 ml离心管，制备1mg / ml的TCO-NHS的（2.7毫摩尔）的无水DMF溶液。 </li><li>在1.7 ml离心管，制备2mg / ml的huA33的（13.3μM）溶液在1ml磷酸盐缓冲盐水，pH 7.4中（0.01M PO 4 3-， </sub&gt; 0.154摩尔NaCl）。 </li><li>使用小的等分试样（&lt;5微升）为0.1M的Na 2 CO 3中 ，调整的抗体溶液的pH至8.8-9.0。请使用pH试纸或pH计用微电极监测pH值，并注意不要让pH值升高pH值高于9.0。 </li><li>一旦抗体溶液是在正确的pH值，添加了对应于活化酯8摩尔当量的TCO-NHS溶液的体积。例如，添加7.9微升1mg / ml的TCO-NHS溶液（1.07×10 -7摩尔的TCO-NHS），以1毫升2毫克/毫升huA33抗体溶液（1.33×10 -8摩尔huA33）。不通过在溶液体积超过5％的DMF。 </li><li>轻轻颠倒的离心管几次混合溶液。 </li><li>包装在不透明的铝箔的离心管中。 </li><li>让溶液孵育1小时，在室温温和搅拌。 </li><li> 1小时在室温之后，使用预填充的一次性SIZ纯化所得免疫Ë排除脱盐柱。首先，如所描述的供应商到储存过程中除去存在于列中的任何防腐剂冲洗尺寸排阻柱。然后，将反应混合物添加到尺寸排阻柱，冲洗用1.5ml 0.9％的无菌盐水的柱，并随后收集用2-毫升0.9％的无菌盐水作为洗脱液的产物。 注:此步骤将产生完成huA33-TCO为2 ml的溶液。 </li><li>测定所得huA33-TCO上的紫外可见分光光度计的浓度。 </li><li>如果浓度较高是理想的，集中使用离心过滤器单元具有50000分子量截止的huA33-TCO溶液。 注意:需要注意的是，虽然huA33和其他各种公知的抗体（ 例如，贝伐单抗，曲妥单抗，西妥昔单抗，和J591）非常宽容被浓缩，凝聚，沉淀可以在其他情况下，会发生在浓度是很重要的。研究人员尝试此procedURE用新的抗体应该相信文献或自身抗体的知识问题方面是否集中的抗体。 </li><li>存储完成huA33-TCO免疫在4°C黑暗环境中。 注意:这是在程序中可接受的停止点。已完成的单抗的TCO偶联物应为这些储存条件下至少3个月是稳定的。 </li></ol> TZ-BN-NOTA 3. 64铜放射性同位素标记注:该协议的这一步涉及的处理和操纵的放射性。在执行这些步骤 - 或进行任何其他工作与放射性 - 研究人员应该与他们的家机构的辐射安全部咨询。采取一切可能的措施，以尽量减少暴露于电离辐射。 <ol><li>在1.7 ml离心管，制备0.5毫克/毫升（723μM）TZ-BN-NOTA的溶液。 </li><li>在一个1.7毫升的MicroCentrifuge管中，加入10微升的TZ-BN-NOTA溶液（5微克）的400微升的0.2M的NH 4 OAC pH值为5.5的缓冲。 </li><li>在适当的放射化学音符维持兴趣，测量和使用剂量校准之前和在下面的协议（3.4-3.8）的随后的步骤后记录样品中放射性的量。这将有助于准确测定放射化学产量。 </li><li>加入2000微居里64铜（74活度）将TZ-BN-NOTA的解决方案。 注意:通常情况下，[64铜]的CuCl 2中的0.1N的HCl的小体积（&lt;30微升）被提供，并且因此只有少量（&lt;10微升）此原料液都需要的放射性标记反应。如果需要更大的[64铜] 氯化铜库存量，放射性标记反应是宽容提高整体反应体积。然而，放射性标记反应溶液的pH值应仔细监测，以确保它不低于pH值4.0。 </li><li>使该溶液温育在室温下10分钟，温和搅拌。 </li><li>之后孵育10分钟，用纯化逆相C 18 HPLC层析产物。保留时间明显高度依赖于每个实验室（泵，柱，管， 等等 ）的高效液相色谱法的设备的设置。然而，到目前的例子中，如果使用梯度5:95的MeCN / H 2 O（均含有0.1％TFA）至95:5的MeCN / H 2 O在15分钟时，64的Cu-Tz-的保留时间BN-NOTA应约为9.8分钟，同时游离的未络合铜64会与溶剂前沿洗脱在约2-4分钟。 </li><li>使用旋转蒸发器，取出的HPLC洗脱剂。 </li><li>再溶解64的Cu-TZ-BN-NOTA产物在0.9％无菌盐水。 注意:由于64 Cu的12.7小时物理半衰期，这不是在程序中一个可接受的停止点。 -NOT的Cu-TZ-Bn的执行器64的合成一个紧接注射放射性的，并遵循立即步骤4.5步骤3.7。 </li></ol> 4. 体内预靶向PET成像注意:由于协议中的第3节，该协议的这一步涉及到处理和操作放射性。在执行这些步骤，研究人员应该与他们的家机构的辐射安全部咨询。采取一切可能的措施，以尽量减少暴露于电离辐射。 <ol><li>在一个雌性无胸腺裸小鼠皮下植入1×10 6 SW1222结肠癌细胞，并允许这些（接种后9-12天），以长成100-150毫米3异种移植物。11 </li><li>稀从协议第2节的huA33-TCO溶液至0.5毫克/毫升的浓度在0.9％无菌盐水的等分试样。 </li><li>注入200微升huA33-TCO溶液（100微克）至尾静脉的异种移植物承载鼠标。 </li><li>允许24小时为huA33-TCO在小鼠的肿瘤积累。 </li><li>稀释64的Cu-TZ-BN-NOTA放射性为1.5毫居里/ ml，在0.9％无菌盐水中的浓度。 </li><li>注入200微升64的Cu-TZ-BN-NOTA放射性溶液（300微居里; 11.1活度; 1.6纳摩尔64的Cu-TZ-BN-NOTA，假设6.7活度/纳摩尔的特定活性）至尾静脉的异种移植物的小鼠。 </li><li>在所需的成像时间点（ 例如，2个，6个，12个，或24小时后喷射），麻醉的小鼠用2％异氟烷:氧混合气。 </li><li>将鼠标放在小动物PET扫描仪的床。氧混合气:使用1％异氟烷在扫描期间保持麻醉。在此之前放置在扫描仪床上的动物，用脚趾捏法验证麻醉和适用兽医药膏鼠​​标，以防止在麻醉过程中干燥的眼睛。 </li><li>通过静态获得为鼠标在PET数据扫描用最少的使用350-700千电子伏的能量窗和6纳秒重合定时窗口20000000同步事件。完成收购的图像后，不要离开鼠标无人看管，不把它关在笼子里与其他老鼠，直到它恢复意识。 </li></ol>

<ol>
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The authors have nothing to disclose.

这个预靶向PET成像策略的主要优点在于，它能够描绘肿瘤靶 - 背景图像的对比度，在通过直接标记的抗体所产生的辐射剂量的一小部分的。例如，在此处描述的结肠直肠癌的成像系统，从急性生物分布实验数据进行了用来执行剂量计算为64 Cu基预靶向策略连同直接标记64的Cu-NOTA-huA33和89锆DFO-huA33。相比，更临床相关的89 Zr的标记的抗体，这些计算清楚地说明了预靶向系统的剂量好处，特别是。有效剂量预靶向策略是0.0124毫西弗/活度，而89锆DFO-huA33超过30倍以上:0.4162毫西弗/活度。比较到64时，预靶向的剂量好处是不太明显的Cu-贴上了ntibody（0.0359毫西弗/活度），虽然有利影响依然存在。 一本IEDDA预靶向方法的最显著优势是它的模块化: 反式 -cyclooctene可以追加到未内化的任何抗体，以及各种各样的货物的可附连到四嗪。事实上，写这个协议我们的主要动机是为了让其他研究小组采用这种方法具有不同的抗体/抗原/放射性同位素系统。沿着这一思路，我们认为关键是要解决一些适应这种方法对其他系统时，研究人员应该考虑的问题。 第一，该抗体的选择是极其重要的。简单地说，该抗体必须是非内在化或内以非常慢的速率。而理想的动力学参数都尚未确定，所述抗体和反应反式 -cyclooctene它携带必须保持在细胞的外部，用于喷射的放射性配体之前，该抗体的内化和隔离将极大地减少体内点击反应的数量。在这里所描述的系统中，huA33抗体目标和结合到A33抗原，表达&gt; 95％的结肠直肠癌的跨膜糖蛋白。重要的是，已经表明，即使在结合其靶，所述huA33抗体/抗原复合物保留在细胞表面为天。31-33虽然是非内在化抗体的必要性是无可否认的限制的策略，一个各种各样的非内化的抗体是已知的，也许是最引人注目的是TAG72靶向CC49抗体ROSSIN 等人已经探索在其优良预靶向的工作。30,34,35 二，本次预定位的策略 - 就像任何其他 - 需要显著优化。除了一个人的身份ntibody和四嗪放射性配，两个关键变量，必须考虑:抗体的注射量和抗体和放射性配体注射之间的间隔时间。我们已经解决了上面的代表结果部这两个变量，但简要地重申，如果有一个太大的抗体或过短的时间间隔中的情况下，显著量的单克隆抗体，缀合物的TCO将保持在血液中，在注射时的放射性。这，反过来，将导致在体内点击结扎发生在血液中，而不是在肿瘤，形成循环，放射性标记的抗体，其将仅缓慢积累在肿瘤随时间。相反，如果任一太少抗体或太长的间隔时间的情况下，放射性的肿瘤的最终量将是次优的。在我们看来，执行严格的成像，或最好，急性生物分布的实验与直接标记抗体ITSELF之前的任何预靶向实验是了解所需的抗体量和初始注射抗体构建体后的理想间隔时间的最可靠的方法。对于不同的注入群众放射性标记的mAb的，这些实验将提供关于放射免疫偶联的两个从血液中的清除率和其在肿瘤蓄积的具体数据，允许的最有前途的条件，预靶向实验选择。 最后，当选择合适的放射性同位素的四嗪基放射性配体的药物动力学必须加以考虑。在这里所描述的系统中，放射性标记的TZ-BN-NOTA部分从主体经由具有约3-4小时的生物半衰期肠道排泄，使得64的Cu的正电子发射放射性同位素具有最互补物理半生活。不幸的是，四嗪基团的生物半衰期太长为它是与第兼容Ë更迅速衰减放射性68嘎（T 1/2 = 68分钟）。在这种情况下，在肿瘤的任何放射性将通过几个半衰期衰变前的多余的放射性配体完成从体内清除。其结果是，图像将具有在早期时间点，当肿瘤-背景活动比率维持低被收购36在理想情况下，后代四嗪放射性会被工程-或许经由PEG化，糖基化，或其它手段-排泄从身体更迅速。这将允许更多的快速衰减的放射性同位素，如68 Ga和18楼这又将进一步增强预靶向成像策略的剂量放射性标记的好处。最终，当研究人员与其他放射性同位素用于成像应用适应这种技术（ 例如 ，124 I，111，（18）F，89的Zr，68镓等 ）或治疗（ 例如， <s向上&gt; 89 Y，177路，225 AC，125 I 等 ），新的四嗪基配位体将需要开发掺入不同螯合剂或放射性标记辅基。这些新型结构的药代动力学的深入调查将是至关重要的，确保配体的间隙性和放射性核素的物理半衰期之间有利的比赛。 最后，我们非常希望其他研究者看到这个预定位技术的承诺，并与新的抗体/抗原系统中使用它。虽然前面的段落说明了这个适应过程可能并不总是简单的，这是我们的信念，这种方法可能对核成像的显著的影响，有针对性的放射性核素治疗，和超越。

在过去的三十年来，正电子发射断层扫描（PET）已经成为在癌症的诊断和管理的一个不可缺少的临床工具。抗体长期以来被认为是有前途的载体用于递送的发射正电子的放射性同位素，以由于其精致的亲和力和特异性的癌症生物标志物的肿瘤。1,2-然而，相对缓慢的体内药物动力学的抗体的要求使用放射性同位素具有多天的物理半衰期。此组合可以产生高辐射剂量的患者的非靶器官，一个重要的并发症，具​​有特别的临床意义，因为放射性免疫静脉内，因此注射​​ - 不像偏身CT扫描 - 结果在吸收剂量在体内的每一个部分，不论审问组织。 为了绕过这个问题，显著的努力一直致力于开发板pment那解耦放射性同位素和靶向部分，从而利用抗体的有利特性，同时避开它们的内在药动学局限性PET成像策略。这些策略-最经常被称为预定位或多步目标 - 通常使用四个步骤:（1）能够结合两个抗原和放射性配体的抗体的施用; （2）在靶组织和从血液中其间隙抗体的积累; （3）小分子放射的管理;和（4） 在体内结扎的放射性配体的抗体，然后用过量的放射性配体的快速清除。3-8。在一些情况下，一个额外的清除剂是步骤2和3之间，以加速的任何抗体的排泄注入即尚未结合肿瘤并保留在血液中。5 从广义上讲，总重量预定位策略的O型是最常见的文献。虽然两人都在临床前模型证明是成功的，他们也拥有了阻碍其临床应用主要限制。第一个策略依赖于链霉亲和标记的抗体和生物素修饰的放射性之间的高亲和性;然而，链霉亲和修饰的抗体的免疫原性已被证明是一个令人担心的问题，相对于平移。5,6,9,10第二种策略，与此相反，采用已基因工程改造以结合两个癌症的双特异性抗体生物标志物抗原和小分子放射性标记的半抗原。3,11-14虽然这后一种途径是肯定的创作，其广泛的适用性是由复杂性，费用，以及缺乏系统的模块化的限制。 最近，我们开发并发布一个预靶向PET成像方法的基础上的逆电子需求狄尔斯 - 阿尔德（我EDDA） 反 -cyclooctene（TCO）和四嗪（Tz的之间的环加成反应;。） 图1 11虽然反应本身就已经知道了几十年，IEDDA化学经历了复兴近年来的点击化学生物耦合技术，就说明了拉尔夫·惠斯勒，约瑟夫·福克斯，和彼得·孔蒂等。12-15 IEDDA环已经在广泛的设置得到应用，包括肽，抗体，和纳米粒子的荧光成像小组的迷人工作以及核成像。既放射性卤素和放射性金属16-26的结扎是高产，清洁，快速（K 1&gt; 30000 M -1秒-1），有选择性的，以及-危重-生物正交27虽然许多类型点击化学的-包括铜催化叠氮炔环加成，应变促进叠氮炔环加成，和施陶丁格LIGations -是生物正交为好，它是快速反应动力学和bioorthogonality，使IEDDA化学所以非常适合在整个生物体的预定位应用的独特组合28,29沿着这条思路，值得注意的是，从最近的报告是非常重要的我们实验室是不是第一个IEDDA化学适用于预靶向:预靶向成像IEDDA的第一份报告源于ROSSIN的工作， 等人 ，配备了SPECT方法采用了111标记嗪30。 如我们上面讨论的，预靶向方法有四个相当简单的步骤（ 图2）。在协议中，在另一方面，一预靶向策略的结肠直肠癌的PET成像，其使用64的Cu-NOTA标记四嗪放射性和huA33抗体的TCO改性共轭进行说明。但是，这种方法的最终模块化是其GR之一eatest资产，如反式 -cyclooctene部分可以被附加到任何非内化抗体，和四嗪可以连接到各种各样的放射性记者的。

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

最初的三个步骤在实验- TZ-BN-NOTA，TCO对huA33缀合的合成，和TZ-BN-NOTA的放射性标记构造（ 图3和4） -是高度可靠的。在上述过程中的情况下，合成在高产率和高纯度的TZ-BN-NOTA结构。所述huA33抗体与4.2±0.6的TCO /单抗进行了修改，并TZ-BN-NOTA是放射性标记的64铜，得到纯化的放射性配体在&gt; 99％放射化学纯度，&gt; 85％的衰变校正收率和比活性〜 6.7活度/纳摩尔（ 图5）。所述huA33-TCO缀合物的活性和四嗪放射性配可以用放射性瞬间薄层色谱法（ITLC）进行测试。 （; 0.55纳摩尔，假定6.7活度/纳摩尔的特定活性100微居里）与稍过量huA33-TCO的;而在PH值，这是通过混合该放射性标记的四嗪进行（50微克0.66纳摩尔）osphate缓冲盐水（pH 7.4）中在室温下5分钟。然后，大约1微居里的溶液的点样到逆相C 18 TLC板上并干燥。该薄层是运行在9:1乙腈:H 2 O，并用放射性TLC板读取器板进行分析。如果点击反应工程按计划进行，结扎64铜NOTA-A33应保持在基准;如果，在另一方面，该反应失败，自由64的Cu-TZ-BN-NOTA将出现在或接近溶剂前沿。 移动到体内成像实验中，在上述协议中，无胸腺裸鼠的A33抗原表达，SW1222结肠癌异种移植物使用。急性的生物分布（N = 5每个时间点）和PET成像（N = 12）的实验表明，预靶向策略能够划定结肠直肠肿瘤的生长具有优良的图像的对比度和高肿瘤-背景的活性比率（ 图6的）。 64的Cu-TZ-BN-NOTA的肿瘤摄取明显在早期时间点:3.5％±0.6％ID /克和4.1％±0.6％ID / g的在1小时和4小时后喷射，分别。然而，在这些早期的点，很容易通过鼠标（肠道遮蔽由放射性结算的量为11.9％±4.4％ID /克和8.8％±3.4％ID / g的在粪便中在1小时和4小时PI，分别）。在几个小时的过程中，多余的放射性配体通过粪便清除（1.4％±0.5％ID / g的在24小时PI），和肿瘤变为在图像（4.0％±0.9％ID / g的在最突出的特点24小时PI）。在这些后来的时间点，肿瘤是公划定的图像中，以及所述肿瘤 - 背景的活性比是相当高;例如，该策略产生肿瘤:26.6±6.6肌肉比率在12小时pi和27.0±7.4在24小时丕不足为奇的是，仅使用64的Cu-TZ-BN-NOTA，非特异性抗体对照实验，邻řhuA33未经缀合的TCO的部分都导致了肿瘤最小摄取。 如将在下面进一步讨论的，这种预靶向策略 - 象所有预靶向策略 - 具有许多变数时，适用于新的抗体/抗原系统，这将需要优化。两个最重要的是单克隆抗体，TCO构建体注射的质量和在注入单抗的TCO构建体并在注射放射性配体之间的间隔的长度。如果单抗-TCO缀合物的量过高或注射之间的间隔时间太短，游离mAb-TCO的血液中的量上升，并出现在血液中，而不是在肿瘤的增大点击反应的可能性。例如，在64铜/ huA33系统在这里所讨论的，为300 huA33（而非100微克）或使用一个12小时的间隔时间（而不是24小时）的微克导致明显增加吨两者施用他量的放射性，在小鼠的心脏（ 图7A和图7B，分别）可见的。在这两种情况下，点击反应仍清楚地发生在肿瘤，如在早期时间点示出由肿瘤摄取的量;然而，放射性标记的抗体在血液中的形成也很明显。虽然这是很有诱惑力的解雇，因为形成于血液中的放射性标记抗体仍然会最终找到自己的方式肿瘤，这有点违背了使用预靶向方法的目的，因为之前到达肿瘤，从而提高了放射性标记抗体会慢慢循环剂量率与非靶器官。相反，如果过少抗体时，吸收在肿瘤的量将自然受到影响。过长的间隔时间也可降低肿瘤摄取的水平慢抗体内化，transcyclooctene异构化，或抗体/抗原脱落的结果。次的诊断ESE问题更有挑战性的，不能简单地实现，通过在PET数据的检查。显然，一个微妙的平衡，必须保持。因此，建议在尝试这一策略应用到新的抗体/抗原系统中的任何研究者使用大量的mAb-TCO构建体（≥200微克）和短间隔时间（≤24小时）作为起点的，并从那里优化。 <img alt="图1" src="/files/ftp_upload/52335/52335fig1.jpg" /> 图1.逆电子点播狄尔斯-阿尔德四嗪和transcyclooctene之间[4 + 2]环加成点击结扎。 <img alt="图2" src="/files/ftp_upload/52335/52335fig2.jpg" /> 图2的四个步骤预靶向方法的一个例子，这个数字是基于最初发表于JNM研究。 Zeglis，BM 等人，一个预靶向PET成像策略浅编辑在bioorthgonal的Diels-Alder反应点击化学。 中华核医学杂志 。54，1389年至1396年（2013年）。 ©2013年核医学和分子成像学会，公司<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52335/52335fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52335/52335fig3.jpg" /> 图3.方案huA33与TCO-NHS的修改。 <img alt="图4" src="/files/ftp_upload/52335/52335fig4.jpg" /> 图4.计划的合成和TZ-BN-NOTA 64铜放射性标记，这个数字是基于最初发表于JNM研究。 Zeglis，BM 等。基于bioorthgonal的Diels-Alder反应一个预靶向PET成像策略点击化学。 中华核医学杂志 。54，1389年至1396年的 （2013年）。 ＆＃169; 2013年核医学和分子成像学会，公司<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52335/52335fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52335/52335fig5highres.jpg" width="500px" /> 图5.纯化的64的Cu-TZ-BN-NOTA的无线型HPLC跟踪。 <img alt="图6" src="/files/ftp_upload/52335/52335fig6.jpg" /> 64铜TZ-BN-NOTA图6 PET图像/ A33-TCO预定位的策略。小鼠皮下移植SW1222（100-150毫米3）经尾静脉注射给药huA33-TCO（100微克）。 24小时后，相同的小鼠施用64的Cu-TZ-BN-NOTA（10.2-12.0活度[275-325微居里]）经尾静脉注射，随后成像的2,6，12，和18小时的给药后放射性药物。 TRansverse（顶部）和冠状（底部）平面图像相交的肿瘤的中心。高水平的吸收在肠道的在早期时间点（ 即，2和6小时）通过12小时很大程度上清楚，而使肿瘤（白色箭头）从所有非靶组织12和18小时后喷射明确划定。这个数字是基于最初发表于JNM研究。 Zeglis，BM 等。基于bioorthgonal的Diels-Alder反应一个预靶向PET成像策略点击化学。 中华核医学杂志 。54，1389年至1396年的 （2013年）。 ©2013年核医学和分子成像学会，公司<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52335/52335fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52335/52335fig7.jpg" /> 图的次优预靶向实验7. PET图像。（A）小鼠BEA环皮下移植SW1222（100-150毫米3，箭头）经尾静脉注射给药huA33-TCO（100微克）。 12小时后，同样的小鼠施用64铜TZ-BN-NOTA（10.2-12.0活度[275-325微居里]）尾静脉注射。 （B）小鼠皮下移植SW1222（100-150毫米3，箭头）经尾静脉注射给药A33-TCO（300微克）。 24小时后，相同的小鼠施用64的Cu-TZ-BN-NOTA（10.2-12.0活度[275-325微居里]）尾静脉注射。在这两种情况下，将小鼠成像注射64的Cu-TZ-BN-NOTA的后12小时。在两个面板，横向（上）和冠状（底部）平面图像相交的肿瘤的中心。而预靶向策略清楚地描述的肿瘤在这两种情况下，相对于与图6显示的结果在这两个图像是不合标 ​​准的。在这两种7A和图7B，是有显著量在心脏背景活动摄取。根据图7A的条件下，这是最有可能的huA33-TCO构造没有被给予足够的时间在肿瘤本地化的结果。根据图7B的情况下，这是可能的注入太多huA33-TCO和具有过量免疫仍然注射后在血液中循环，甚至24小时的结果。这个数字是基于最初发表于JNM研究。 Zeglis，BM 等。基于bioorthgonal的Diels-Alder反应一个预靶向PET成像策略点击化学。 中华核医学杂志 。54，1389年至1396年的 （2013年）。 2013年核医学和分子成像学会，公司<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52335/52335fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

利用生物正交反电子需求的Diels-Alder环的预靶向PET成像

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.7K 次观看.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 

视频: Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

高分辨率光纤内窥镜为原位细胞成像

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

科研

生物工程

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

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 ...

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.

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 ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

测量超极化 [1-13C]硫化的旋转-莱迪兹松弛磁场依赖性

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

利用生物正交反电子需求的Diels-Alder环的预靶向PET成像

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低温固定化细胞跟踪和成像病毒蛋白相关结构的相关光电子显微镜 (克莱)

测量超极化 [1-¹³C]硫化的旋转-莱迪兹松弛磁场依赖性

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

血流成像与超快多普勒