由美国国立卫生研究院这项工作的部分支持授予NIBIB EB002807和CA177744（GRE和SSE）和P41 EB002034到GRE，霍华德·J·哈尔彭，PI，和丹佛大学表示感谢。马克Tseytlin是由美国国立卫生研究院R21 EB022775，NIH K25 EB016040，美国国立卫生研究院/ NIGMS U54GM104942支持。作者感谢瓦列里Khramtsov，现在在西弗吉尼亚大学，并Illirian Dhimitruka在俄亥俄州立大学的pH敏感TAM自由基的合成，以及杰拉德·罗森和约瑟夫·高锟在马里兰大学的mHCTPO合成，PROXYL，BMPO和nitronyl自由基。

1.在250 MHz的快速扫描线圈驱动的安装<ol><li> 快速扫描实验条件的计算 注意:在RS-EPR的最重要的参数是扫描速度，α，这是扫描频率和扫描宽度（式3）的产物。对于窄扫描宽度，更快的扫描速率被使用，并且对于更广泛的扫描宽度，使用较慢的扫描速率。下面的说明步骤通过后一种情况，并展示如何在700万吨扫描宽度和6.8 kHz的扫描频率的实验线圈驱动器的参数到达。 <ol><li>确定谐振器的带宽（BW RES）。 <img alt="式（1）" src="/files/ftp_upload/54068/54068eq1.jpg" /> （1） 其中v 水库是谐振器的工作频率，Q是质量因子。 Q = 90，是用来获取代表性的成果数据的快速扫描谐振器常见。 </li><li>确定的快速扫描速度，α，所有由谐振器带宽欠<img alt="公式（2）" src="/files/ftp_upload/54068/54068eq2.jpg" /> （2） <img alt="公式3" src="/files/ftp_upload/54068/54068eq3.jpg" /> 其中N是常保守选择为5-6的常数，ΔB 页是以mT的峰-峰衍生物线宽，a是如果T / s的洛伦兹线宽的扫描速率。 注:在典型区段的自由基的共同价值是<img alt="公式4" src="/files/ftp_upload/54068/54068eq4.jpg" /> = 0.1 mT的。在早期快速扫描文学比较;方程2通过将信号带宽（BW SIG）等于BW RES而得。 </li><li>确定由速度所允许的最大的快速扫描频率。 <img alt="公式5" src="/files/ftp_upload/54068/54068eq5.jpg" /> （3） <img alt="公式6" src="/files/ftp_upload/54068/54068eq6.jpg" /> 其中w是SCA的宽度n和f是扫描频率。 700万吨的扫描宽度将覆盖频谱的100％ 用于体内电流探头。使用该值，并在（式2）计算出的速度，以确定扫描频率。 <img alt="公式7" src="/files/ftp_upload/54068/54068eq7.jpg" /></li></ol></li><li> 调谐电容器的选择和快速扫描线圈驱动器的调 注意:快速扫描线圈驱动器典型地在一个共鸣模式产生正弦波运行。共振发生在扫描频率，其中电感和电容的电抗是相等的幅度和相反的符号，从而使总电抗是接近于零。 <ol><li>确定使用的电感L的快速扫描线圈在1.1.3确定的频率，和（公式4）适当的电容。 <img alt="式（8）" src="/files/ftp_upload/54068/54068eq8.jpg" /> <img alt="公式9" src="/files/ftp_upload/54068/54068eq9.jpg" /></li><li>从（公式4）在半分割ÇTOT得到电容值用于线圈驱动电容器盒的每一侧。 <img alt="10式" src="/files/ftp_upload/54068/54068eq10.jpg" /> <img alt="11式" src="/files/ftp_upload/54068/54068eq11.jpg" /> 注意:快速扫描线圈驱动器有两个放大器。当选择一个电容器，需要电容器框与上框中的每一侧相等的电容平衡。双方都在系列。 </li><li>拧电容箱的顶盖，两侧是等于在步骤1.2.2确定的值插入电容。 </li><li>更换电容箱的顶部，拧下来，以确保它岿然不动。 </li><li>使用谐振的线圈驱动器的前面板上，调整输出频率，直到正弦波形具有最大幅值。 </li></ol></li></ol> 2.试剂和鬼怪的制备<ol><li> 拉迪的制备CALS <ol><li>从冰箱中取出15 N-PDT和允许容器来室温（10-15分钟）。 </li><li>用分析天平称出1.4毫克的15 N-PDT的。 </li><li>添加1.4毫克15 N-PDT的至15ml去离子（DI）H 2 O为0.5mM的最终浓度。 注意:4-氧代-2,2,6,6-四（2 H 3）甲基-1-（3,3,5,5- 2 H 4，1- 15 N）piperdinyloxyl（15 N-PDT）， 4- 1 H-3-氨基甲酰基2,2,5,5-四（2 H 3）甲基-3- pyrrolinyloxyl（15 N-二mHCTPO）和3-羧基- 2,2,5,5-四（2- H 3）甲基-1-（3,4,4- 2 H 3，1- 15 N），吡咯烷（15 N-二PROXYL）25（ 图1E-G）的基团具有在水溶液中的长期稳定性（2年）和在室温下。其固体形式通常存储在冷冻器或冰箱保持这些基团稳定多年。氮氧自由基的稳定性通常使它们无毒，并且它们的制备可在一个正常的台式完成时的溶剂是水。当使用有机溶剂，在通风橱内准备氮氧解决方案，同时以适当的个人防护装备（PPE）配备。 </li></ol></li><li> pH敏感的三苯甲基自由基的制备 <ol><li>称取0.7毫克三芳基甲基自由基（ATAM 4）26自由基（1,400克/摩尔）和200微升无水乙醇溶解。 </li><li>权衡0.00681克KH 2 PO 4（136.1克/摩尔）和50毫升去离子水中溶解为1mM的终浓度。 </li><li>称量2.8克KOH（56克/摩尔）和50毫升去离子水中的溶解1 M的最终浓度</li><li>添加KOH逐滴向磷酸盐缓冲液（2.2.2）调节为7.0的pH值。 </li><li>加1 mM磷酸盐缓冲液800微升，并在无水乙醇200微升ATAM 4为一个最终浓度在80:20缓冲0.5毫米的entration:乙醇。 </li><li>重复步骤2.2.1-2.2.5建立在pH = 7.2 ATAM 4样本。 </li><li>放置ATAM 4，pH值= 7.0和ATAM 4，pH值= 7.2到单独6毫米石英样品管中。 </li><li>既6毫米石英EPR管置于16毫米石英EPR管，在两者之间有2毫米厚的泡沫塑料垫片。 注意:在石英样品管的壁是厚0.5mm时，并且除了2mm的间隔得到ATAM样品之间3毫米的分离。所使用的pH敏感的三苯甲基自由基在俄亥俄州立大学26合成。这是用于成像的例子被称为ATAM 4。占的pH值敏感性的反应示于图1A。 </li></ol></li><li> BMPO-OH的生成 <ol><li>称出680毫克KH 2 PO 4和100毫升去离子水中溶解为50mM的终浓度。 </li><li>加入1M的KOH中逐滴向磷酸缓冲液至pH = 7.3。 </li><li>称取50毫克BMPO的（199.25克/摩尔）。 </li><li>结合50mg的BMPO与在一个16毫米石英照射管5毫升磷酸盐缓冲液中。 </li><li>加入100微升300毫摩尔的过氧化氢。 </li><li>照射在16毫米的石英照射管中的混合物用中压450瓦特的UV灯5分钟。 </li><li>使用玻璃移液管，转移2.5毫升照射BMPO-OH溶液中，并从石英照射管并进入具有3 mm分频器一个16毫米石英样品管的一侧。 </li><li>传输剩余2.5毫升照射BMPO-OH的成具有3 mm分频器和16毫米的石英样品管的另一侧。 </li></ol></li><li> dinitroxide激进的制备 <ol><li>称取24.7毫克2H，15 N -二硫化物dinitroxide（ 图1C）在1ml DMSO中为47.5毫米的储备溶液。 </li><li>制备的10mM Tris缓冲液，并调整pH至7.2。 </li><li>就拿40微升dinitroxide原液并用T​​ris缓冲液稀释至1mM的终浓度。 </li><li>放入缓冲液250微升dinitroxide解决方案在16毫米石英样品管在中心10毫米分频器。 </li><li>称出154毫克的谷胱甘肽和添加到5ml Tris缓冲液为100mM的终浓度。 </li><li>加上10mm的分配器的一侧5微升的100mM谷胱甘肽溶液250微升1mM的dinitroxide溶液的双基转化为单价。 </li></ol></li><li> 氮氧自由基的制备 <ol><li>从冰箱中取出基和允许容器来室温（10-15分钟）。 </li><li>称取1.9毫克nitronyl（390克/摩尔）。 </li><li>称取0.56毫克KOH的和在10毫升去离子水中溶解为1mM的终浓度。 </li><li>混合1.9毫克nitronyl的到10ml 1mM的KOH溶液0.5毫nitronyl的终浓度。 注意:如果necess元，使用涡旋或超声发生器的nitronyl的速度溶解。 </li></ol></li></ol>在250 MHz的快速扫描仪3.安装注意:与氮氧自由基的水溶液样品，这对谐振器Q和调谐作为缓冲溶液类似的效果，共振器的调谐是设置为样本被成像的好方法<ol><li> 调整与氮氧自由基的水样品的谐振器。 <ol><li>插入水样品中的15毫升中含0.5毫15 N-PDT成16毫米石英EPR管。 </li><li>插入石英管进入交叉循环的RS-EPR谐振器的检测侧。 </li><li>改变乐器源的频率，直到它包含示例的检测侧的频率相匹配。通过在软件中输入所需的值手动更改250MHz的源的载波频率。 </li><li>改变激励侧的频率，以匹配frequen谐振器的实验源和检测侧的CIES。通过根据制造商的协议的谐振腔内转动的可变电容器改变励磁侧的频率。 </li></ol></li><li> 设置仪器控制台和主磁体 <ol><li>打开光谱仪，并选择随时间横轴上的记录瞬时数据的实验。 </li><li>内的软件，设置点到65,536的数目和时间基准10纳秒。 </li><li>设置平均次数为10,000一个强大的或窄的信号，并以45,000广泛的或弱信号。 </li><li>按下软件中的"接触"按钮，从软件发送的实验参数到控制台和激励的主要领域磁铁。 </li><li>主磁场设为9 mT的。 </li><li>将电源旋钮衰减到50分贝，打开7瓦大功率功放。 </li></ol></li></ol> 4.执行快速扫描实验注意:有关含有BMPO-OH 24幻影分析的具体说明，pH敏感TAM自由基19,27和氧化还原敏感dinitroxides 28在文献中提供。 <ol style="margin-left: 40px;"><li> 标准氮氧样品的功率饱和 注意:有利的是做在其上将用于看自由基至pH或氧化还原状态敏感相同的实验条件下，一个标准的氮氧游离基样本的功率饱和曲线。 <ol><li>打开快速扫描线圈驱动，从第1节（6.8千赫兹的扫描频率和700万吨的扫描宽度）的值。 </li><li>在50分贝开始，收集了快速扫描光谱与10万的平均水平。减少3分贝的衰减和重复测量。继续直到0 dB的衰减器设置，或只要在桥上读出的隔离测量&lt;0。 </li><li>传输ŧ他生快速扫描数据转换成卷积程序（例如写在Matlab）和处理原始数据转换为吸收光谱。 </li><li>输入扫描频率，扫描宽度，分，时基数量进入该程序，并运行程序来处理原始快速扫描信号转换成吸收信号。 </li><li>积的吸收信号的振幅作为平方根功率（以瓦特）入射到谐振器的功能。在非饱和制度，振幅为线性依赖于入射功率的平方根。 </li><li>适应趋势线开始于0,0和包括落入线性响应区域的所有数据点。线性响应区域，正比于微波功率的平方根信号振幅增加。 </li><li>推断这种趋势将更高的权力，并比较EPR信号强度。对于使用该信号幅度不会从外推趋势线偏离超过3％的最高权力。在奥德r表示的快速扫描信号的卷积能够正常工作，该信号仍然必须在线性响应区域相对于入射功率。 注:原快速扫描数据的传输可以通过网络连接或通过拇指驱动器来完成。在这种情况下，传输是必要的，因为程序来处理原始数据（Matlab的）并不是具有数据收集软件在同一台计算机上。其处理的原始数据的反卷积算法29进行说明。 </li></ol></li></ol>

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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=Improved+Sensitivity+for+Imaging+Spin+Trapped+Hydroxyl+Radical+at+250+MHz.">Improved Sensitivity for Imaging Spin Trapped Hydroxyl Radical at 250 MHz.</a> Chem. Phys. Chem. 16 (3), 528-531 (2015).</li><li>Burks, S. R., Bakhshai, M. A., Makowsky, M. A., Muralidharan, S., Tsai, P., Rosen, G. M., Kao, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2H,+15N-Substituted+nitroxides+as+sensitive+probes+for+electron+paramagnetic+resonance+imaging.">2H, 15N-Substituted nitroxides as sensitive probes for electron paramagnetic resonance imaging.</a> J. Org. Chem. 75, 6463-6467 (2010).</li><li>Dhimitruka, I., Bobko, A. A., Hadad, C. M., Zweier, J. L., Khramtsov, V. V. <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+characterization+of+amino+derivatives+of+persistent+trityl+radicals+as+dual+function+pH+and+oxygen+paramagnetic+probes.">Synthesis and characterization of amino derivatives of persistent trityl radicals as dual function pH and oxygen paramagnetic probes.</a> J. Am. Chem. Soc. 130 (32), 10780-10787 (2008).</li><li>Elajaili, H. B., 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=Electron+spin+relaxation+times+and+rapid+scan+EPR+imaging+of+pH-sensitive+amino-substituted+trityl+radicals.">Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals.</a> Magn. Reson. Chem. 53 (4), 280-284 (2015).</li><li>Elajaili, H., Biller, J. R., Rosen, G. M., Kao, J. P. Y., Tseytlin, M., Buchanan, L. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isotopic+labeling+of+a+nitroxide+spin+label+to+enhance+sensitivity+for+T2+oxymetry.">Selective isotopic labeling of a nitroxide spin label to enhance sensitivity for T2 oxymetry.</a> J. Magn. Reson. 90, 40-51 (1990).</li><li>Tsai, P., 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=Esters+of+5-carboxyl-5-methyl-1-pyrroline+N-oxide:+A+family+of+spin+traps+for+superoxide.">Esters of 5-carboxyl-5-methyl-1-pyrroline N-oxide: A family of spin traps for superoxide.</a> J. Org. Chem. 68 (20), 7811-7817 (2003).</li><li>Biller, J. 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=Frequency+dependence+of+electron+spin+relaxation+times+in+aqueous+solution+for+a+nitronyl+nitroxide+radical+and+perdeuterated-tempone+between+250+MHz+and+34+GHz.">Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz.</a> J. Magn. Reson. 225, 52-57 (2012).</li><li>Rosen, G. 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=Dendrimeric-containing+nitronyl+nitroxides+as+spin+traps+for+nitric+oxide:+Ssynthesis,+kinetic,+and+stability+studies.">Dendrimeric-containing nitronyl nitroxides as spin traps for nitric oxide: Ssynthesis, kinetic, and stability studies.</a> Macromolecules. 36 (4), 1021-1027 (2003).</li><li>Bobko, A. 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=Redox-sensitive+mechanism+of+no+scavenging+by+nitronyl+nitroxides.">Redox-sensitive mechanism of no scavenging by nitronyl nitroxides.</a> Free Radical Biol. Med. 36 (2), 248-258 (2004).</li><li>Roshchupkina, G. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+EPR+measurement+of+glutathione+in+tumor-bearing+mice+using+improved+disulfide+biradical.">In vivo EPR measurement of glutathione in tumor-bearing mice using improved disulfide biradical.</a> Free Radical Bio. Med. 45 (3), 312-320 (2008).</li><li>Mitchell, D. 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=Use+of+Rapid-Scan+EPR+to+Improve+Detection+Sensitivity+for+Spin-Trapped+Radicals.">Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals.</a> Biophysical Journal. 105 (2), 338-342 (2013).</li><li>Bobko, A. A., Dhimitruka, I., Zweier, J. L., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trityl+radicals+as+persistent+dual+function+pH+and+oxygen+probes+for+in+vivo+electron+paramagnetic+resonance+spectroscopy+and+imaging:+Concept+and+experiment.">Trityl radicals as persistent dual function pH and oxygen probes for in vivo electron paramagnetic resonance spectroscopy and imaging: Concept and experiment.</a> J. Am. Chem. Soc. 129 (23), (2007).</li><li>Biller, J. 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=Electron+spin-lattice+relaxation+mechanisms+of+rapidly-tumbling+nitroxide+radicals.">Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals.</a> J. Magn. Reson. 236, 47-56 (2013).</li><li>Redler, G., Barth, E. D., Bauer, K. S., Kao, J. P. Y., Rosen, G. M., Halpern, H. J. <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+electron+paramagnetic+resonance+imaging+of+differential+tumor+targeting+using+cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.">In vivo electron paramagnetic resonance imaging of differential tumor targeting using cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.</a> Magn. Reson. Med. 71 (4), 1650-1656 (2013).</li></ol>

We have nothing to disclose.

快速扫描信号具有比CW较高频率分量，并要求根据线宽，弛豫时间较大谐振器的带宽，和快速扫描的速度。对于给定的实验所需要的带宽是基于所述线宽和磁场（式2）的扫描速率。取决于探头的弛豫时间进行研究（T 2和T 2 *），和扫描速度，振荡可以出现在该信号的后沿。对于氮氧自由基和T 2〜500纳秒在250兆赫（ 第 57 届洛矶山会议磁共振，EPEL，B 等 ，2015），实验的扫描率往往不高到足以观察到任何振荡。 实验带宽通常由谐振器的带宽限制。快速扫描实验的每半个周期记录有要么降低或增加场/频率，所以实验带宽是半吨他谐振器的带宽，如图（等式1）。这样，它是如果实验带宽由参数的选择的限制比谐振器的带宽更大的振荡被衰减，拓宽在反褶积线的结果。由于实验带宽由速率和自由基的线宽测定正在研究，理解这些特征是快速扫描实验的一个关键组成部分。 目前的协议在250 MHz的探头含有氧气，粘度，pH值，内源性的瞬态信号分子敏感的幻影（ 即 ，OH•，NO•）和氧化还原状态表明EPRI。 1到3mm之间的空间分辨率已经证明，与实验获得乘以29秒（2线15 N光谱的单线， 图3）和15分钟之间（的全谱为5μMBMPO-OH， 图5）。方法开发幻影表演使用RS-EPR图像取代传统的CW-EPR成像技术23,24，并打开使用EPR的探针在活体成像的新途径。 EPRI是基于对荧光或磷光其他体内成像技术是有利的，如EPR探针是对更广泛的体内现象敏感。此外，在250兆赫的RF穿透〜7厘米，以便在更深的层次异常组织进行研究。核磁共振成像（MRI）提供了非常详细的解剖图，但斗争提供量化的生理信息。 MRI和EPRI的组合有朝一日结果在正电子发射断层扫描（PET）/计算机断层扫描（CT）扫描器的所有磁共振版本。这样的文书将提供PET / CT的同样的好处，但没有沉重的辐射剂量或昂贵的无线电示踪剂。 方法开发幻影继续推动Ť他限制了RS-EPR的，但最终的目标是实现在利用动物模型实验室的技术。用于图像重建的计算将需要加以改进，以加速数据收集为一个4D实验（3空间，1光谱维）。一种改进的算法，目前正在开发的，并且是在体内应用必不可少的，然而原则的证据可以与2D成像来完成。 许多原子团，如15 N-PDT，在体模中使用的快速降解体内仅为60秒半衰期的条件下。与在体内减少39的改进的抵抗自由基已被合成，并且是在体内建立足够大的浓度非常重要。 RS-EPR过CW-EPR 24的增强的灵敏度将是解决这一问题的另一个好处。快速扫描的灵敏度是目前为5μM的幻象，并为100μM和5mM之间，这取决于探针将被成像，在芝加哥大学正在执行动物研究（个人通信，马吉欧，M。，2015）。该方法RS将继续发展，以弥补这一差距，但应用程序已经开始进入实际的体内应用 （ 第 57落基山会议磁共振，EPEL，B， 等人 ，2015年）。

相对于其他医疗成像模态，电子顺磁共振成像（EPRI）是唯一能够定量图像生理特性包括扩散的pH值1-3，PO + 2 4-7温度8，灌注和组织9的生存能力，微粘度和易于小分子10和氧化应激11。在组织谷胱甘肽（GSH）和细胞12,13便于二硫化物断开的估计可以在氧化还原状态报告。 对于体内成像，EPR在250 MHz和1GHz的频率范围内选择，因为这些频率提供组织穿透的足够的深度（最多几个厘米），以产生用于小动物，其中强度不被介电损耗的效果降低的图像。更高的频率，例如9.5 GHz的14（X波段）和17千兆赫（KÚ波段）15,16可用于皮肤和头发或单个细胞的成像， 分别。 EPRI的所有频率的成功取决于顺磁自旋探针是特异性的组织，使它们的位置和命运可能被成像。 如果电子自旋探头的环境空间异质性的EPR谱的所有位置的总和。谱空间成像将样本的数量成小空间段的排列并计算每个段17的EPR谱。这允许通过测量EPR谱图的空间变化的局部环境的映射。磁场梯度用于编码空间信息成EPR谱，这是所谓的突起。谱空间图像从这些突起18,19重建。 的RS-EPR的磁场是在一个时间，是相对于电子自旋弛豫时间短（ 图2）20,21通过共振扫描。 ð<html>快速扫描信号的econvolution给出吸收光谱，这相当于以往的一阶导数的CW频谱的第一积分。快速扫描信号在正交检测，以使自旋系统响应的吸收和分散组分进行测量。这实质上是收集每单位时间的数据量的两倍。在快速扫描实验的信号饱和发生在比连续波更高的功率，所以更高的功率可以不为饱和关注使用。20,22许多更平均每单位时间相比，CW进行。更高的功率，直接正交检波和每单位时间更多的平均结合，得到快速扫描一个更好的信噪比（SNR），特别是在限定的空间分离的高梯度的预测，从而导致更高质量的图像。为了达到所需的约10倍左右长CW作为快速扫描23幻像的图像相同的SNR。 帐篷"&gt;信噪比增加还允许实验在250 MHz的被OH的反应与形成低浓度自旋阱加合物5-叔丁氧羰基-5-甲基-1- pyrroline- N-氧化物（BMPO-OH），这将是具有二硫化物接头连接不可见的顺时针方法24。Dinitroxides是谷胱甘肽的切割敏感，因此可以对细胞的氧化还原状态的报告。平衡存在，取决于本谷胱甘肽的浓度时，二-和单-自由基形式之间。观察这些变化需要整个5 mT的宽光谱的捕获，并且可以比在一个连续试验步进磁场与快速扫描的EPR快得多实现。 一个完整的快速扫描系统由四个部分组成:光谱仪，主磁场磁体，快速扫描线圈驱动器，并迅速扫描跨环谐振器。分光计和主磁场磁体功能中的相同的CW实验中，设置主塞曼字段和收集从谐振器中的数据。快速扫描线圈驱动器产生进入的快速扫描跨环谐振专门设计的快速扫描线圈正弦扫描电流。对快速扫描的跨环谐振器的快速扫描的线圈产生大的均匀的磁场，这是在频率3和15千赫之间扫过。

<table border="0" cellpadding="0" cellspacing="0" width="1763">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4,1-15N)piperdinyloxyl (15N PDT)</td>
			<td style="width:152px;">CDN Isotopes&#160;</td>
			<td style="width:135px;">M-2327</td>
			<td style="width:964px;">98% atom 15N, 98 % atom D, Quebec Canada</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4-1H-3-carbamoyl-2,2,5,5-tetra(2H3)methyl-3-pyrrolinyloxyl (15N mHCTPO)</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in Reference 29</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">3-carboxy-2,2,5,5-tetra(2H3)methyl-1-(3,4,4-2H3,1-15N)pyrrolidinyloxyl (15N Proxyl)</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">707-SQ-100M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4)piperdinyloxyl (14N PDT)</td>
			<td style="width:152px;">CDN Isotopes</td>
			<td style="width:135px;">D-2328</td>
			<td>98% atom D, Quebec Canada</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">pH sensitive trityl radical (aTAM4)</td>
			<td style="width:152px;">Ohio State University</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at Ohio State University and described in reference 26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Potassum Phosphate, Monobasic</td>
			<td style="width:152px;">J.T. Baker Chemicals</td>
			<td style="width:135px;">1-3246</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">6 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">Q-5M-6M-0-250/RB</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">8 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">Q-7M-8M-0-250/RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)&#160;</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Hydrogen Peroxide</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:135px;">H1009 SIGMA</td>
			<td>30%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">16 mm Quartz EPR tube</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">16-7PP-11QTZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Medium Pressure 450 W UV lamp</td>
			<td style="width:152px;">Hanovia</td>
			<td style="width:135px;">679-A36</td>
			<td>Fairfield, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">L-Glutathione, reduced</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:135px;">G470-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Nitronyl</td>
			<td style="width:152px;">NA</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 31</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Sodium Hydroxide&#160;</td>
			<td style="width:152px;">J.T. Baker Chemicals</td>
			<td style="width:135px;">1-3146</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid scan electron paramagnetic resonance opens new avenues for imaging physiologically important parameters in vivo

我们使用快速扫描电子顺磁性共振（RS-EPR），其可以在体内条件下提供对氧浓度，pH值，氧化还原的定量信息，在250兆赫表明稳定的自由基报道分子的2D谱空间成像的优良的方法状态和信号分子的浓度（ 即，OH•，NO•）。将RS-EPR技术具有更高的灵敏度，改进的空间分辨率（1毫米），并且相比于标准的连续波（CW）技术更短的采集时间。各种假想的配置已经过测试，与空间分辨率改变从1到6毫米，报道分子范围从16μT（160毫克）到5 mT的（50克）的频谱宽度。跨环双峰谐振器解耦激发和检测，降低了噪音，而快速扫描效应允许更多的电力被输入到饱和之前自旋系统，增加的EPR信号。这个导致相当高的信噪比比常规的CW EPR实验。

实验的产物是一组被重建成二维（一个频谱，一个空间）图像用假色标来表示信号幅度的突起。深蓝色表示基线的地方没有信号，绿色是低振幅和红色最高。沿着x轴（光谱尺寸）片描绘在磁场轴的EPR信号（EPR转变）。沿着y轴（空间尺寸），信号之间的分离对应于谐振器的样本之间的物理空间上的分离。 图3示出了两个图像，以连续波（ 图3B）或具有三种不同类型的15 N取代的氮氧自由基（ 图3D）幻像的RS（ 图3A）获得的比较。最广泛的信号对应于15 N-PROXYL，五元吡咯啶环与在生理pH下带负电荷，这可能有助于靶向分子于特定细胞区室。所述双峰信号属于15 N-mHCTPO和是一个单一的氢之中否则完全氘化的结果。这种单一的分割进行了优化，监测氧浓度30的变化。最窄的信号来自15 N-PDT，这完全是一个氘灵活哌啶环。它可用于监测的氧浓度，或氧化还原环境（减少结构导致在EPR信号降低）。 对于相同的5分钟采集时间中，RS图像显示每个基团的频谱图案的优良的空间分辨率和清晰度。用于RS超过CW的改进的一个原因可通过在两种技术（ 图3C）之间的两个不同的梯度强度相比较的光谱可以看出。随着梯度强度增大频谱信号变宽。高梯度（100万吨/平方厘米）的编码空间信息下的连续频谱的相当恶化。 因为一个导数信号比吸收信号变宽更迅速，信噪比为最高梯度CW突起（红色迹线）相比，最高的梯度的RS凸起（蓝色迹线）的非常差。线宽为空间位置的函数可以从一个二维曲线图中提取。线宽将根据氧浓度或粘度周围的氮氧探头的变化是宽或窄。在图3A中摄像的虚线是在室温和开放到空气中。因为氧含量和粘度（由温度测定）保持稳定，每个探针的线宽应是跨越含有自由基每个管的宽度恒定。 图4示出了线宽的散布从片适合通过比较该2D图像至真线宽值（黑色水平线）。图像切片值，尤其是对15 N-PDT，是比CW（ 图4B）更好的匹配，为RS（ 图4A）的真实线宽值。这也是RS在CW技术的改进的SNR的结果。 将RS技术的另一个好处是在很短的时间，以产生宽磁均匀场扫描的能力。在250 MHz的实验一个典型的扫描频率为9 kHz，对应0.11毫秒。这是0.11毫秒的场扫描是0.5 MT或5.0 mT的。与此相比，CW，其中5.0 mT的扫描需要几十秒到几分钟。快速扫描就能够迅速收集的光谱信息100％在时间，这是适合于体内成像。 图5展示范围广泛的RS-EPR我maging应用于自旋捕捉模式。重要的信号分子，如OH•和•NO内源性自由基有很短的寿命。为了研究这些分子，"自旋捕获"被使用。自旋阱31（BMPO）用OH•的反应的一个例子示于图1B。含5μMBMPO-OH加成物幻影成像如图5（A，B）。自旋阱加合物信号是依赖于OH•的起始浓度和具有30分钟，以便让这产生OH•任何进程的研究的半衰期。所述nitronyl nitoxide 32被用作广谱成像的另一个例子，但在过去已被用于NO•33,34的自旋捕获。含有nitronyl幻像成像示于图5（C，D）。对于SP在陷阱，捕获整个频谱允许原始的临时自由基，这是目前较好的称号。 敏感性像pH值和氧化还原状态的生理变化是从整个光谱的变化的。 图6显示成像ATAM 4。在图6B中，ATAM 4在pH轮廓= 7.0（蓝色）具有许多的光谱特征，和从图像切片与相应的零梯度谱（绿色）匹配良好。在pH = 7.4， 图6C与相应的零梯度谱一致比较这ATAM 4的轮廓，用较少的光谱特征和静止。含在它的二聚体的dinitroxide的幻影成像，并减少单体形式示于图7中 ，两个不同的谱通过二硫键（SS）的裂解产生的，所以传送灵敏度氧化还原的environment 1,35。 <img alt="图1" src="/files/ftp_upload/54068/54068fig1.jpg" /> 图1. EPR探针对许多生理变化敏感。（A）中的pH敏感三-芳基甲基（TAM）自由基26的一个例子。 （B）旋转陷阱BMPO。 （C）15 N-dinitroxide。 （D）的nitronyl。 （E）15 N-PROXYL。 （F）15 N-mHCTPO。 （G）15 N-PDT。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54068/54068fig2.jpg" /> 图2. 快速扫描EPR具有固有更好的信噪比。（A）我ÑCW EPR振幅h是总信号的一小部分，由磁场调制来确定。 （B）在直接检测的快速扫描，检测在整个信号幅度。噪声增大的信号是在由大肠杆菌产生的超氧实验明显肠球菌是在X波段被困BMPO。出于同样的30秒采集时间，几乎没有任何信号，而在一个强烈的信号在快速扫描光谱（D）36观察到的CW频谱（C）观测。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig2large.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/54068/54068fig3.jpg" /> 图3. 信噪比提高允许更好的空间分辨率。对于同一个5分钟的采集时间，遥感图像（ <st荣&gt; A）具有更高的信噪比和比较，一个与CW（B）获得的空间分辨率。 （ 三 ）有与快速扫描（蓝色）和CW（红色）收购的预测具有较好的一致性时，没有梯度的存在（0 MT /厘米）（D）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54068/54068fig4.jpg" /> 图4. 快速扫描图像的信息量要比CW高。（A）二维遥感图像的条带。二维图像CW（ 二 ）切片。每个样品的真实线宽（黑色水平线）示出了用于比较。见参考文献23。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig4large.jpg" target="_blank">请点击这里</a><html>查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54068/54068fig5.jpg" /> 图5. 快速场扫允许在几秒钟的整个频谱的捕获。（A）中的2D的谱空间由BMPO-OH加合物的幻象的图像。 （B）模拟适合在250 MHz的零梯度BMPO-OH谱拟合初始BMPO-OH图像和含BMPO-OH区域和含噪声的区域之间进行区分。 （℃）14 N nitronyl基团，其可用于在体内的一氧化氮的俘获。通过每个谱（D）的片显示出在250兆赫的频谱形状。见参考文献19。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ve_content"FO: - together.within页保留="1"&gt; <img alt="图6" src="/files/ftp_upload/54068/54068fig6.jpg" /> 图6. 频谱的任何部分留出来，从而更好地监测生理引起的光谱变化。（A）二维光谱空间图像包括pH敏感ATAM 4激进的两管的幻象。 （B）在pH ATAM 4的光谱轮廓= 7.0（蓝色）和相应的零梯度谱（绿色）。 （℃）在pH ATAM 4的光谱轮廓= 7.4 B（蓝）和相应的零梯度谱（绿色）。见参考文献19,26,37。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig6large.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/54068/54068fig7.jpg" /> 图7。 </st荣&gt; 快速扫描打开大门， 在250 MHz的体内氧化还原的监测。15 N-dinitroxide的（A）的二维谱空间图像。 （ 二 ）通过顶部（蓝色线）和底部（红色曲线）车厢中的两个图像切片。 （C）的顶部隔室保持不变，但底部隔室已经减少与谷胱甘肽。 （D）的切片通过每个图像对象表示所述底部隔室的一维谱的变化。见参考文献1,28,35。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54068/54068fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo at JoVE.com

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

新的电子顺磁共振（EPR）方法，快速扫描EPR（RS-EPR），则展示了其优于传统的连续波（CW）技术，打开了活体成像的新场地的二维光谱的空间影像。结果，在250 MHz的被证实，但该技术可应用于任何频率。

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 ...

rapid-scan-electron-paramagnetic-resonance-opens-new-avenues-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

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

8.5K 次观看.  University of Denver. 新的电子顺磁共振（EPR）方法，快速扫描EPR（RS-EPR），则展示了其优于传统的连续波（CW）技术，打开了活体成像的新场地的二维光谱的空间影像。结果，在250 MHz的被证实，但该技术可应用于任何频率。 

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

内镜下黏膜下注射材料性能评价的一种新的体内模型

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

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 ...