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>
	<li>Wu, 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=Antibodies+and+antimatter:+The+resurgence+of+immuno-PET.">Antibodies and antimatter: The resurgence of immuno-PET.</a> Journal of Nuclear Medicine. 50, 2-5 (2009).</li><li>Zeglis, B. M., Lewis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+the+construction+of+radiometallated+bioconjugates+for+positron+emission+tomography.">A practical guide to the construction of radiometallated bioconjugates for positron emission tomography.</a> Dalton Transactions. 40, 6168-6195 (2011).</li><li>Hollander, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies+for+cancer+therapy.">Bispecific antibodies for cancer therapy.</a> Immunotherapy. 1, 211-222 (2009).</li><li>Liu, 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=Tumor+pretargeting+in+mice+using+99mTc-labeled+morpholino,+a+DNA+analog.">Tumor pretargeting in mice using 99mTc-labeled morpholino, a DNA analog.</a> Journal of Nuclear Medicine. 43, 384-391 (2002).</li><li>Boerman, O. C., van Schaijk, F. G., Oyen, W. J. G., Corstens, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeted+radioimmunotherapy+of+cancer:+Progress+step+by+step.">Pretargeted radioimmunotherapy of cancer: Progress step by step.</a> Journal of Nuclear Medicine. 44, 400-411 (2003).</li><li>Goldenberg, D. M., Sharkey, R. M., Paganelli, G., Barbet, J., Chatal, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+pretargeting+advances+cancer+radioimmunodetection+and+radioimmunotherapy.">Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.</a> Journal of Clinical Oncology. 24, 823-834 (2006).</li><li>Sharkey, R. M., Chang, C. H., Rossi, E. A., McBride, W. J., Goldenberg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretargeting:+taking+an+alternate+route+for+localizing+radionuclides.">Pretargeting: taking an alternate route for localizing radionuclides.</a> Tumor Biology. 33, 591-600 (2012).</li><li>Sharkey, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+delivery+of+radionuclides+for+imaging+and+therapy+of+cancer+using+pretargeting+methods.">Improving the delivery of radionuclides for imaging and therapy of cancer using pretargeting methods.</a> Clinical Cancer Research. 11, 7109-7121 (2005).</li><li>Schultz, 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=A+tetravalent+single-chain+antibody-streptavidin+fusion+protein+for+pretargeted+lymphoma+therapy.">A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.</a> Cancer Research. 60, 6663-6669 (2000).</li><li>Lewis, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+evaluation+of+pretargeted+64Cu+for+tumor+imaging+and+therapy.">In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.</a> Journal of Nuclear Medicine. 44, 1284-1292 (2003).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pretargeted+PET+imaging+strategy+based+on+bioorthgonal+Diels-Alder+click+chemistry.">A pretargeted PET imaging strategy based on bioorthgonal Diels-Alder click chemistry.</a> Journal of Nuclear Medicine. 54, 1389-1396 (2013).</li><li>Blackman, M. L., Royzen, M., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine+ligation:+fast+bioconjugation+based+on+inverse+electron+demand+Diels-Alder+reactivity.">Tetrazine ligation: fast bioconjugation based on inverse electron demand Diels-Alder reactivity.</a> Journal of the American Chemical Society. 130, 13518-13519 (2008).</li><li>Devaraj, N. K., Upadhyay, R., Hatin, J. B., Hilderbrand, S. A., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+pretargeted+labeling+of+cancer+cells+through+a+tetrazine/trans-cyclooctene+cycloaddition.">Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.</a> Angewandte Chemie-International Edition. 48, 7013-7016 (2009).</li><li>Devaraj, N. K., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+tetrazine+cycloadditions.">Biomedical applications of tetrazine cycloadditions.</a> Accounts of Chemical Research. 44, 816-827 (2011).</li><li>Devaraj, N. K., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-based+cycloadditions:+application+to+pretargeted+live+cell+imaging.">Tetrazine-based cycloadditions: application to pretargeted live cell imaging.</a> Bioconjugate Chemistry. 19, 2297-2299 (2008).</li><li>Keliher, E. J., Reiner, T., Turetsky, A., Hilderbrand, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yielding,+two-step+18F+labeling+strategy+for+18F-PARP1+inhibitors.">High-yielding, two-step 18F labeling strategy for 18F-PARP1 inhibitors.</a> ChemMedChem. 6, 424-427 (2011).</li><li>Reiner, T., Earley, S., Turetsky, A., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+small-molecule+ligands+for+PARP1+imaging+in+living+cells.">Bioorthogonal small-molecule ligands for PARP1 imaging in living cells.</a> ChemBioChem. 11, 2375-2377 (2010).</li><li>Reiner, T., Keliher, E. J., Earley, S., Marinelli, B., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+in+vivo+imaging+of+a+18F-labeled+PARP1+inhibitor+using+a+chemically+orthogonal+scavenger-assisted+high-performance+method.">Synthesis and in vivo imaging of a 18F-labeled PARP1 inhibitor using a chemically orthogonal scavenger-assisted high-performance method.</a> Angewandte Chemie International Edition. 50, 1922-1925 (2011).</li><li>Taylor, M. T., Blackman, M., Dmitrenko, O., Fox, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+highly+reactive+dienophiles+for+the+tetrazine-trans-cyclooctene+ligation.">Design and synthesis of highly reactive dienophiles for the tetrazine-trans-cyclooctene ligation.</a> Journal of the American Chemical Society. 133, 9646-9649 (2011).</li><li>Selvaraj, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+integrin+alpha(v)beta(3)+targeted+PET+tracer+based+on+a+cyclic+RGD+peptide.">Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin alpha(v)beta(3) targeted PET tracer based on a cyclic RGD peptide.</a> Bioorganic and Medicinal Chemistry Letters. 21 (3), 5011-5014 (2011).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+18F+labeling+of+cysteine-containing+peptides+and+proteins+using+tetrazine-trans-cyclooctene+ligation.">Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.</a> Molecular Imaging. 12, 121-128 (2013).</li><li>Han, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+bioorthogonal+and+highly+efficient+conjugation+method+for+quantum+dots+using+tetrazine-norbornene+cycloaddition.">Development of a bioorthogonal and highly efficient conjugation method for quantum dots using tetrazine-norbornene cycloaddition.</a> Journal of the American Chemical Society. 132, 7838-7839 (2010).</li><li>Zeglis, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+strategy+for+the+construction+of+radiometalated+antibodies+for+positron+emission+tomography+based+on+inverse+electron+demand+Diels-Alder+click+chemistry.">Modular strategy for the construction of radiometalated antibodies for positron emission tomography based on inverse electron demand Diels-Alder click chemistry.</a> Bioconjugate Chemistry. 22, 2048-2059 (2011).</li><li>Zeng, D., Zeglis, B. M., Lewis, J. S., Anderson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+impact+of+bioorthogonal+click+chemistry+on+the+development+of+radiopharmaceuticals.">The growing impact of bioorthogonal click chemistry on the development of radiopharmaceuticals.</a> Journal of Nuclear Medicine. 54, 829-832 (2013).</li><li>Reiner, T., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inverse+electron+demand+Diels-Alder+reaction+in+radiochemistry.">The inverse electron demand Diels-Alder reaction in radiochemistry.</a> Journal of Labeled Compounds and Radiopharmaceuticals. 57, 285-290 (2014).</li><li>Li, Z., 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=Tetrazine-trans-cyclooctene+ligation+for+the+rapid+construction+of+18-F+labeled+probes.">Tetrazine-trans-cyclooctene ligation for the rapid construction of 18-F labeled probes.</a> Chemical Communications. 46, 8043-8045 (2010).</li><li>Karver, M. R., Weissleder, R., Hilderbrand, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+series+of+1,2,4,5-tetrazines+for+bioorthogonal+conjugation.">Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation.</a> Bioconjugate Chemistry. 22, 2263-2270 (2011).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+chemistry:+fishing+for+selectivity+in+a+sea+of+functionality.">Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.</a> Angewandte Chemie International Edition. 48, 6973-6998 (2009).</li><li>Bosch, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+strained+alkynes+for+Cu-free+click+reaction+in+live+mice.">Evaluation of strained alkynes for Cu-free click reaction in live mice.</a> Nuclear Medicine and Biology. 40, 415-423 (2013).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+chemisry+for+pretargeted+tumor+imaging+in+live+mice.">In vivo chemisry for pretargeted tumor imaging in live mice.</a> Angewandte Chemie International Edition. 49, 3375-3378 (2010).</li><li>Ackerman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, 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=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., vanden Bosch, S. M., LaForest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+reaction+for+tumor+pretargeting:+In+vivo+chemistry+can+boost+tumor+radiation+dose+compared+with+directly+labeled+antibody.">Diels-Alder reaction for tumor pretargeting: In vivo chemistry can boost tumor radiation dose compared with directly labeled antibody.</a> Journal of Nuclear Medicine. 54, 1989-1995 (2013).</li><li>Rossin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

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?...

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...

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

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

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&#039; 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 ...

科研

生物工程

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 ...

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 ...

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 ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

为生物医学和生物工程研究建立章鱼生态系统

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

芳香族氨基酸调控细胞代谢的生物正交化学成像

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

虚拟现实交互分析 Zebrafish 心脏收缩

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

作者聚焦：斑马鱼中的高分辨率 4D 光片成像和虚拟现实用于心脏功能的单细胞分析

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

斑马鱼心脏收缩的 4D 光片成像

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...