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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<li>Biller, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22J.+Magn.+Reson%22%5BJournal%5D)">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/pubmed?term=((Dendrimeric-containing+nitronyl+nitroxides+as+spin+traps+for+nitric+oxide%3A+Ssynthesis%2C+kinetic%2C+and+stability+studies%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">Dendrimeric-containing nitronyl nitroxides as spin traps for nitric oxide: Ssynthesis, kinetic, and stability studies.</a> Macromolecules. 36 (4), 1021-1027 (2003).</li>
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<li>Mitchell, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Rapid-Scan+EPR+to+Improve+Detection+Sensitivity+for+Spin-Trapped+Radicals%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">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/pubmed?term=((Trityl+radicals+as+persistent+dual+function+pH+and+oxygen+probes+for+in+vivo+electron+paramagnetic+resonance+spectroscopy+and+imaging%3A+Concept+and+experiment%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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/pubmed?term=((Electron+spin-lattice+relaxation+mechanisms+of+rapidly-tumbling+nitroxide+radicals%5BTitle%5D)+AND+%22J.+Magn.+Reson%22%5BJournal%5D)">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/pubmed?term=((In+vivo+electron+paramagnetic+resonance+imaging+of+differential+tumor+targeting+using+cis-3%2C4-di%28acetoxymethoxycarbonyl%29-2%2C2%2C5%2C5-tetramethyl-1-pyrrolidinyloxyl%5BTitle%5D)+AND+%22Magn.+Reson.+Med%22%5BJournal%5D)">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</em...

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><tbody><tr><td>4-oxo-2,2,6,6-tetra(&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;3&lt;/sub&gt;)methyl-1-(3,3,5,5-&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;4&lt;/sub&gt;,1-&lt;sup&gt;15&lt;/sup&gt;N)piperdinyloxyl (&lt;sup&gt;15&lt;/sup&gt;N-PDT)</td><td>CDN Isotopes&amp;nbsp;</td><td>M-2327</td><td>98% atom &lt;sup&gt;15&lt;/sup&gt;N, 98 % atom D, Quebec Canada</td></tr><tr><td>4-&lt;sup&gt;1&lt;/sup&gt;H-3-carbamoyl-2,2,5,5-tetra(&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;3&lt;/sub&gt;)methyl-3-pyrrolinyloxyl (&lt;sup&gt;15&lt;/sup&gt;N-mHCTPO)</td><td>N/A</td><td>N/A</td><td>Synthesized at U. Maryland and described in Reference 29</td></tr><tr><td>3-carboxy-2,2,5,5-tetra(&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;3&lt;/sub&gt;)methyl-1-(3,4,4-&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;3&lt;/sub&gt;,1-&lt;sup&gt;15&lt;/sup&gt;N)pyrrolidinyloxyl (&lt;sup&gt;15&lt;/sup&gt;N-Proxyl)</td><td>N/A</td><td>N/A</td><td>Synthesized at U. Maryland and described in Reference 25</td></tr><tr><td>4 mm Quartz EPR Tubes</td><td>Wilmad Glass</td><td>707-SQ-100M</td><td></td></tr><tr><td>4-oxo-2,2,6,6-tetra(&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;3&lt;/sub&gt;)methyl-1-(3,3,5,5-&lt;sup&gt;2&lt;/sup&gt;H&lt;sub&gt;4&lt;/sub&gt;)piperdinyloxyl (&lt;sup&gt;14&lt;/sup&gt;N-PDT)</td><td>CDN Isotopes</td><td>D-2328</td><td>98% atom D, Quebec Canada</td></tr><tr><td>pH sensitive trityl radical (aTAM&lt;sub&gt;4&lt;/sub&gt;)</td><td>Ohio State University</td><td>N/A</td><td>Synthesized at Ohio State University and described in Reference 26</td></tr><tr><td>Potassum Phosphate, Monobasic</td><td>J.T. Baker Chemicals</td><td>1-3246</td><td></td></tr><tr><td>6 mm Quartz EPR Tubes</td><td>Wilmad Glass</td><td>Q-5M-6M-0-250/RB</td><td></td></tr><tr><td>8 mm Quartz EPR Tubes</td><td>Wilmad Glass</td><td>Q-7M-8M-0-250/RB</td><td></td></tr><tr><td>5-tert-butoxycarbonyl-5-methyl-1-pyrroline-&lt;em&gt;N&lt;/em&gt;-oxide (BMPO)</td><td>N/A</td><td>N/A</td><td>Synthesized at U. Maryland and described in Reference 30</td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma Aldrich</td><td>H1009 SIGMA</td><td>30%</td></tr><tr><td>16 mm Quartz EPR tube</td><td>Wilmad Glass</td><td>16-7PP-11QTZ</td><td></td></tr><tr><td>Medium Pressure 450 W UV lamp</td><td>Hanovia</td><td>679-A36</td><td>Fairfield, NJ</td></tr><tr><td>L-Glutathione, reduced</td><td>Sigma Aldrich</td><td>G470-5</td><td></td></tr><tr><td>Nitronyl</td><td>NA</td><td>N/A</td><td>Synthesized at U. Maryland and described in Reference 31</td></tr><tr><td>Sodium Hydroxide&amp;nbsp;</td><td>J.T. Baker Chemicals</td><td>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?...

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 ...

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Research

JoVE Journal

Bioengineering

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

9.4K 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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

超极化<sup&gt; 13</sup&gt;ç代谢磁共振波谱与成像

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

癌症研究

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

一个多腔动态单一酶幻影超极化磁共振剂研究的用途

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

工程学

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

电子顺磁共振在生物样品环境温度和 77 k 的应用

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

生物化学

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

无创在体内小动物的MRI和MRS：基本实验程序

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

生物学

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

神经科学

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

医学

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

电子自旋共振氧测绘活体物种显微成像

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

快速扫描电子顺磁共振打开新的途径成像生理重要参数

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