E.K. gratefully acknowledges the support of the Graduate School of Bioengineering (GSB) at Technische Universit&#228;t M&#252;nchen. This work was supported by the German Research Foundation (DFG) within the SFB Collaborative Research Center 824, "Imaging for Selection, Monitoring, and Individualization of Cancer Therapies."

1.样品原液制备<ol><li> gadoterate葡甲胺（GadM，0.5摩尔/升）加入到浓缩的[1- 13 C]丙酮酸得到1-毫摩尔/升GadM的终浓度。添加三苯甲基自由基三（8-羧基-2,2,6,6-四（羟基乙基）-benzo- [1,2-4,5] - 双 - （1,3）-dithiole -4-基） - 盐甲基钠（OX063）到该混合物中，得到15毫摩尔/ L的终浓度。涡旋直至完全溶解。 注意:此储备溶液制备被设计用于与3.35-T临床前的DNP超极化的使用。当使用7-T临床超极化，不需要gadoterate葡甲胺，因为，在更高的磁场，它的好处是可以忽略不计。加入含钆造影剂的增加实现的固态极化，也是极化率。然而，在液体状态下，造影剂缩短的T 1弛豫时间。 </li></ol> 2.成长的细胞培养<ol><l我&gt;成长PC3细胞在培养瓶中有125 平方厘米的增长领域。使用含有10％胎牛血清（FCS）的F-12K培养基，并维持在37℃下将细胞在湿润气氛下，在5％的CO 2。溶解步骤之前，请从培养瓶中的媒介。 注:每个细胞需要细胞传播特定的制备方案。咨询与细胞线路提供商的要求。 </li></ol> 3.将细胞用于实验的制备<ol><li>除去细胞培养基和洗涤与细胞〜10毫升的磷酸盐缓冲盐水（PBS）的。 </li><li>加入5毫升的胰蛋白酶到烧瓶中，并在细胞培养瓶返回到培养箱3至5分钟。 </li><li>添加〜5毫升的F-12K培养基的停用胰蛋白酶。 </li><li>计数使用自动细胞计数器细胞。混合用染色液10μL的细胞溶液10μL。用吸管拌匀传送混合液10μL到第一个"计数玻璃"电子室。 </li><li>除去并在该烧瓶（多个）计数细胞。转移含有细胞的期望数量的适当的卷（ 例如，5×10 6至10 8）到塑料瓶中。 </li><li>离心将细胞在1200×g离心5分钟并弃上清。 </li><li>重悬在含有10％FCS，以800微升的总体积中的F-12K培养基中的细胞，并将它们传送到反应杯（2毫升）中。放置反应杯到塑料瓶中装满温水。 </li></ol> 4.溶解剂配制注:溶解剂是用来溶解的超极化样品的液体。在生物应用中，溶解通常用H 2 O类或氧化氘（D 2 O）系缓冲剂，如PBS或三（羟甲基）氨基甲烷（TRIS）进行的，含有1克/升乙二胺四乙酸（EDTA）。 <ol><li> Prepara20毫摩尔/升的PBS缓冲液和灰<ol><li>制备100毫升的溶解剂，溶解36毫克磷酸二氢钠（的NaH 2 PO 4），247毫克磷酸二钠（ 磷酸氢二钠），并且在20毫摩尔氢氧化钠/升钠溶液10毫克的EDTA（氢氧化钠）的D 2 O.混合妥善直至完全溶解。 注:EDTA（1克/升）被添加到缓冲器，以消除可能的铁磁离子，其可以破坏超极化。该NaOH用于中和在一个1的丙酮酸:1摩尔比以达到pH为7.4。 </li></ol></li></ol> 5.变温插杆（VTI）冷却时间<ol><li>在DNP-NMR偏振片程序主窗口中，单击"冷却"。 注意:此接通真空泵和抽空VTI至大约5.0毫巴。随后，VTI和液态氦容器之间针阀完全打开，允许液体氦流入VTI。流速为r通过针阀egulated保持在VTI液态氦的最优量，直到它到达氦沸腾温度。然后，VTI被抽空到几乎完全真空，并在温度达到约1.4 K的VTI充满液体氦高达65％。此时，该仪器是准备样品插入。 </li></ol> 6.样品制备和插入<ol><li>用微量中，加入13 C标记的样品原液〜8μL到一个塑料杯。 </li><li>将塑料杯的插入杆，并开始按在主程序窗口"插入样品"的样品插入过程。选择"正常样本"，然后点击"继续"。 注意:在此过程中，针阀先关闭，以停止液体氦的流量为VTI，并在VTI的压力则增大。在VTI内的样品支架从液体氦，入口阀凸在VTI的顶部打开，气态氦流从入口阀引入，以防止空气中的水分外界污染。 </li><li>出现提示时，推插杆与连接的塑料杯，下入VTI。确保在VTI底部到达样品架。否则，气态氦可以推动样品从VTI的。 </li><li>分离并取出插入杆。 </li><li>点击"下一步"，在对话窗口中完成该过程。样品插入过程不应该需要更长的时间超过10秒。 注:入口阀然后关闭，气态氦流中断时，与样品杯的样品架被浸入液态氦和针阀打开以允许液体氦流入VTI。后5 - 10分钟时，VTI被冷却到低于1.4 K，使所有的自由电子被极化。 </li><li>确认该塑料杯用样品通过检查吨正确地引入到VTI帽子它不附着到插入杆或氦气从VTI推出。然后点击"完成"。 </li></ol> 7.微波扫描（可选） 注:微波扫频允许最佳的微波频率的决心最大化目标化合物的13 C核的超极化率。 <ol><li>为了测量微波扫描，启动RINMR程序，键入"HYPERSENSENMR"，然后点击"选择配置"和"待办事项Microsweep"。 </li><li>要启动过程中，选择程序主窗口中的"校准"选项卡。 </li><li>点击"生成"，选择开始和结束频率（ 例如，94.100千兆赫-94.200千兆赫）时，频率步长（ 例如，20兆赫）时，电源（100毫瓦），和时间（60秒）。点击"继续"，"启用"和"开始"。 注意:使用这些设置，超极化第一极化样本使用的94.100千兆赫的微波频率和100mW的功率60秒。然后，它适用于一个90°的射频（RF）脉冲，并使用内置的分光计获得超极化13 C信号。反复在指定的频率范围的每个步骤，这些步骤。对于后续的超极化，选择与实测的最大信号幅度的微波频率。 </li></ol> 8.极化<ol><li>为了测量极化集结，开始RINMR程序，键入"HYPERSENSENMR"，然后点击"选择配置"和"固体堆积。" </li><li>在DNP-NMR偏振片程序主窗口中，单击"两极分化"启动超极化过程。 </li><li>选择最佳的微波频率（微波扫描期间获得）和样品的功率（ 如 100毫瓦），然后单击"下一步"。 </li><li>启用"两极分化集结监控"，然后单击"完成"。 </li><li>极化样品&gt; 95％（〜60分钟为[1- 13 C]丙酮酸盐）。 注意:在偏振，微波被导入VTI和到样品，从而导致13 C-旋转到与超极化不成对电子自旋对齐。为了测量超极化积累，具有5°的翻转角（FA）RF脉冲，周期性地施加（ 例如，每300秒），并将所得的信号被绘制为一个偏振积聚曲线。 </li></ol> 9.解散<ol><li>当极化达到&gt; 95％，通过点击DNP-NMR偏振片程序主窗口中的"解散"启动溶解过程。 </li><li>从下拉菜单中选择溶解过程，然后单击"下一步"。 注:偏振器允许一个通过选择追气体的定时来定义所期望的溶解过程。 </li><li>负载〜5毫升通过顶部阀门的溶解剂，成吨的加热的容器偏振片的他溶解一部分。计算的溶解剂的确切量使用以下等式需要: <img alt="式（1）" src="/files/ftp_upload/54751/54751eq1.jpg" /> 其中V DA为溶解剂中，M PA，男OX063的所需体积，以及m 迦是丙酮酸群众，OX063和gadoterate葡甲胺，分别添加到样品储液。 </li><li>将溶解粘在进气门上方的主动地位。 注意:这允许仪器解散仪器连接到在VTI样品杯。 </li><li>点击"完成"开始溶解过程。 注:溶解剂由氦气加压到3巴，并随后加热至200℃，引起压力的增加。当压力达到10巴时，针形阀关闭，以停止液氦流入VTI。样品架引发杯从液态氦。溶解棒下降到VTI和连接到样品杯中。该空调溶解剂由压力，从而从含有溶解缓冲液和氦气，通过溶解粘在杯加热容器产生推压，使所述样品的快速溶解。该溶液然后流出到通过塑料管的收集烧瓶。与所连接的杯溶解棒然后从VTI上升。 </li><li>移动溶解粘在杯附加的"清洁"的位置，并通过点击完成该过程"完成"。 </li></ol> 10. 13 C超极化信号的检测<ol><li> 在体外 13 C代谢磁共振波谱<ol><li>混合从与细胞溶液800微升的收集烧瓶20毫摩尔/升溶解的超极化样品的200微升。 注:最终得到的浓度的[1- 13 C]丙酮酸是4毫摩尔/升。 </li><li>混合使用公微量和转让〜600微升到5毫米NMR管中的悬浮液。 </li><li>插入5毫米NMR管到1-T的NMR波谱仪。在软件的主窗口中，单击"运行"开始测量，应用系列，每3是一百10°RF脉冲。 注:测量与细胞超极化样品的初始混合和分光采集的开始之间的时间。确保混合过程不超过30秒，以减少极化损失。 </li></ol></li><li> 13 C代谢磁共振成像<ol><li>要建立使用MRI谱仪的体外实验容器，取5毫升注射器，并将其连接到一个导管（D = 1.2 MM）是足够长的距离光谱仪的ISO为中心，以达到的平易近人区光谱仪。 </li><li>装满次体外容器为实验所需浓度电子的细胞溶液（ 例如，10 8），或与酶溶液。 </li><li>放置一个体外容器在MRI磁体的等角点。放置在容器上的13 C-调谐射频接收器线圈。放置一个集中13 C标记定位模型（ 例如，10 mol / L的13 C尿素）附近。 </li><li>插入" 体外容器"傍核磁共振扫描器的等中心。 </li><li>运行扫描仪的标准的3平面定位序列和调节体外容器的位置向异中心，根据需要。 </li><li>运行1小时T2加权"解剖"涵盖体外容器位序列。使用以下设置:二维自旋回波具有轴向取向，重复时间（TR）= 2000毫秒，回声时间（TE）= 20毫秒，层厚= 1毫米，视图覆盖在体外容器字段，和每激发16相呼应。确保场匀场在这一步上质子完成。 </li><li>在解剖图像中，选择中心感兴趣区域5个连续切片。开一个13 C光谱校准收购覆盖所选的解剖切片。使用以下设置:二维块西格特校准序列与轴向方向12×12为中心进行编码，TR = 1000毫秒，层厚= 5毫米，视图匹配解剖图像，扫描（NS）= 64，带宽的数目字段= 5000赫兹，和FA = 90°。 </li><li>选择13 C光谱校准序列（有关详细信息，请参阅舒尔特等 2011），来自75个脉冲序列库。点击下载脉冲序列从计算机扫描仪"下载"。点击"光谱预扫描"，以运行光谱预扫描。在频谱幅度图，调整从13 C校准幻影峰到CENTE扫描频率河设置接收机增益为最大。点击"开始"，以运行13 C光谱校准序列。注意报告发送增益和中心频率。 </li><li>设置13 C化学位移成像（CSI）收购覆盖所选的解剖切片。使用以下设置:二维回波平面波谱成像（EPSI）具有轴向取向12×12为中心进行编码，TR = 400毫秒，层厚= 5毫米，视图匹配解剖图像的领域中，NS = 300，和带宽= 5,000赫兹。 注:EPSI样品单行在k-空间之后，将重复一个RF激励同时获取空间和光谱信息。有关收购技术的详细信息，请参阅德斯特等人的文章。 2015年76。 </li><li>下载13 C CSI序列和运行光谱预扫描。调整扫描仪的频率和由校准序列指定发送的增益输出。 </li><li>超极化溶液沉积在收集烧瓶后，制定〜3毫升到注射器，然后将其注入到连接到体外的容器的导管。开始采集。采集完成后，保存用于后续的重建的原始数据文件。 </li></ol></li></ol> 11.数据重构<ol><li>应用两个所述动力学模型中的一个来分析所获取的数据。 <ol><li>在用于说明的LDH动力学第一方法中，动力学值（k）时，乳酸盐信号（M LAC）的总和相比较的所有超极化分子的信号（M x）的 21,77。 <img alt="公式（2）" src="/files/ftp_upload/54751/54751eq2.jpg" /></li><li>在另一种方法，测量随着时间的推移乳酸和丙酮酸信号，将它们装配到动力学模型17,25,71。为了解决代谢汇率，K PA→LAC，有效乳酸，R LAC的信号衰减率，使用通过两个现场交流差分模式，产生乳酸下面的线性微分方程: <img alt="公式3" src="/files/ftp_upload/54751/54751eq3.jpg" /></li></ol></li></ol>注:有效乳酸盐信号衰减率r 的LAC是依赖于乳酸纵向弛豫时间（T 1，LAC），相反的代谢汇率从乳酸为丙酮酸ķLAC→PA，施加的FA和TR以及的信号强度丙酮酸（M PA）和乳酸（M LAC），同时考虑到每个相继激励后的不可逆信号还原: <img alt="公式4" src="/files/ftp_upload/54751/54751eq4.jpg" />因此，R LAC导致信号衰减的单，形影不离的术语。由于可以校正在t他翻转角和重复时间，即使是有磁通LAC→PA，我们假定从乳酸为丙酮酸（K LAC→PA）的汇率不需要被包括在计算的基础上，其结果Harrison 等。 2012 78。他们的研究结果显示，第k LAC→PA不玩为一体会承担至关重要的作用。该模式允许量化乳酸的T 1弛豫时间。这个模型是独立丙酮酸施用到测量，其中，在体外实验的情况下，并不重要，并且可以忽略不计。它，然而，起到用于在体内测量79中起重要作用。

Rolf F. Schulte and Marion I. Menzel are employed with GE Global Research.

13 CMRSI与超极化探针是实时监测代谢的体外 和体内有前途的方法。其中很重要的一个方面，当使用这种实验过程是适当的标准化，体外实验特别是有关。首先，需要正确地和一致地完成，以实现每个实验超极化材料的相同浓度试样的制备。这需要一个精确称重两种样品是超极化和缓冲。如果浓度是不正确的，该溶液的最终pH是不准确的，它可以有对T 1的细胞的...

目前，为肿瘤分期，再分期，治疗反应监测，复发的检测多种癌症的最广泛使用的临床方法是[18 F] -FDG的PET。 [1]然而，最近，一些新颖的方式和其他方式已经出现。其中的一个方法是13 CMRSI。该技术包括引入13 C-分子成的生物样品，接着微创MRI评估在体外或在实时体内的代谢。尽管如此，13 CMRSI的最大的挑战，相对于其他方法，如[18 F] -FDG的PET或计算机断层扫描，是它的低信噪比。 NMR信号成正比偏振在两个能态的自旋1/2核人口差与总人口的比例（ 图1A）的水平。偏振是第一个产品细胞核电子旋磁比（γ）和在温度所施加的磁场强度。的1 H核的典型极化是在0.001％至0.005％的顺序在3 T，它给出了一个相对较差的信噪比。今天的国家的最先进的MRI是一个成功的成像方法，不仅是因为高丰1小时生物样品中和1小时的高旋磁比（γ= 1H兆赫42.576 / T）。然而，观察其他核，如碳，更苛刻。唯一稳定的，磁活性碳同位素，13℃，只占1.1％的所有碳原子。此外，13℃（γ13C = 10.705兆赫/ T）的旋磁比大于1小时的低四倍，从而导致更低的检测效率。总之，低13 C丰度和低γ13C引起热13 C测量来实现的一个1的灵敏度的0.0176％H-NMR测定体内 。 动态核极化 克服13 C测量相对较差的灵敏度的方法是DNP。它是由阿尔伯特·W·欧沃豪斯对金属的最初描述于1953年。在他的文章中，他指出:"这表明，如果传导电子的电子自旋共振是饱和的，细胞核将被极化，以相同的程度，如果他们的旋磁比为使电子自旋的，他们将是"2后来那年，卡弗和Slichter实验证实的Overhauser的假设3。 1958年，Abragam和宝洁描述了这种效应在液体电子和它命名为"坚实的效果。"在温度低于4 K，电子自旋极化达到近100％，是大小比核自旋极化（ 图1B）4更高的超过三个数量级。 ŧ发生了由于电子的旋磁比（γE = 28024.944兆赫/ T）为大小比核旋磁比更高的三个数量级。电子和原子核，如Overhauser效应，固体效果，交叉效果，热混合效果之间的弱相互作用，允许极化的传递使用微波照射以频率靠近相应的电子从电子自旋核自旋顺磁共振（EPR）频率5,6。 DNP理论得到了进一步发展涉及到更多的电子和热混合。然而，迄今为止，DNP的不统一的量化理论描述已发表7,8。 <img alt="图1" src="/files/ftp_upload/54751/54751fig1.jpg" /> 图1:了解动态核极化和超极化。 A）自旋人口的示意图对比在热平衡偏振状态和超极化的状态。 B）中的偏振取决于温度。 （E - ）的电子的极化达到低于1.4 K的DNP 100％允许偏振从电子到13 C核，这增加了它们的偏振高达10 5倍的转移。 <a href="http://ecsource.jove.com/files/ftp_upload/54751/54751fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在使用13 C-NMR生物系统的研究介绍DNP，随后快速样品溶解已被开发。 50年代以后的Overhauser的假设，扬H. Ardenkjaer拉森等人。解决使超极化冷冻样品到液体状态以最小的超极化损失6的技术上具有挑战性的问题。解散DNP打开一个新的研究领域被称为13 CMRS我，提供了新的方法来研究和描述各种疾病状态9,10。不成对电子，三苯甲基自由基三（8-羧基-2,2,6,6-四（羟基乙基）-benzo- [1,2-4,5] - 双 - （1,3），作为稳定的载体-dithiole -4-基）盐 - 甲基钠（OX063）或（2,2,6,6-四甲基哌啶-1-基）氧基（TEMPO）通常被使用。这些都与期望的13 C标记的分子混合并暴露于微波辐射与接近相应的EPR频率的频率。使用这种技术的13 C核的极化可以增加高达37％11。这导致了10 5倍极化增强相比，热平衡极化11,12。然而，只要在微波辐射停止和/或13 C-分子转移到液体状态，极化与已偏振的13 C核的纵向弛豫时间（T 1）衰减。因此，该快速溶解技术发明或任何后续技术缩短实验测量（ 即注射）之前是生物应用13关键的时间。 有迹象表明，候选分子需要满足成功13 CMRSI研究三大要求。第一，所关心的13 C核必须具有足够长的T 1（&gt; 10秒）。的13 C-标签的选择是至关重要的。最佳候选细胞核碳经由键用1 H-细胞核没有直接接触。它也需要在2被迅速代谢- 3个T 1次，导致下游代谢产物与从原始物质的显著不同的化学位移。样品混合物还必须形成无定形固体状态时玻璃，使得空间分布减小电子和13 C之间的距离，从而使反两极分化的外汇储备。如果候选分子不自然地形成非晶化玻璃，它需要在装配玻璃剂高度可溶的，如甘油或二甲基亚砜14。这些要求导致相对小数目的候选分子。然而，即使是合适的分子的成功发现后，开发工作的协议为超极化可以是技术上具有挑战性9,14,15。 近年来，一些衬底已成功偏振光，如[1- 13 C]丙酮酸12,16 - 36，[2- 13 C]丙酮酸37，[1- 13 C]丙酮酸乙酯38，[1- 13 C ]乳酸39，[1- 13 C]富马酸40 - 43，13 C-碳酸氢盐36,44,45，[1- 13 C]乙酸钠43,46 - 49，13 C尿素6,36,50,51 ，[5- 13 C] glutamiNE 15,52,53，[1- 13 C]谷氨酸53,54，[1- 13 C] 2-酮戊二酸55，[1- 13 C]丙氨酸，和其他人14,56。对于超极化一个特别有趣的和常用的基材是[1- 13 C]丙酮酸盐。它被广泛用于临床前研究以调查在各种疾病14,17,22细胞能量代谢。 [1- 13 C]丙酮酸满足所有成功超极化的要求，包括随后被代谢前穿过细胞膜相对长T 1和快速运输。用[1- 13 C]丙酮酸临床前研究，目前正在翻译成诊所57。 丙酮酸代谢 众所周知，有一种癌细胞的DNA，并在其代谢途径变化突变之间的直接联系。早在20世纪20年代，奥托华宝discovERED有葡萄糖和生产乳酸的肿瘤中增加的代谢相比健康组织58 - 60。接着，在其他代谢途径，如戊糖磷酸途径，三羧酸循环，氧化磷酸化，和核苷酸和脂类的合成各种替换，已经描述。 丙酮酸是糖酵解的终产物。在肿瘤，它经历由乳酸脱氢酶61催化的无氧酵解和与辅酶烟酰胺腺嘌呤二核苷酸的还原形式反应 （NADH），产生乳酸和辅酶（NAD +）的氧化形式。可替代地，丙酮酸经过与谷氨酸以形成丙氨酸氨基转移反应，由丙氨酸转氨酶（ALT）的催化。这两种反应是可逆的。丙酮酸也经历脱羧通过丙酮酸脱氢酶（PDH）催化二氧化碳和乙酰-CoA河epresenting在该步骤的不可逆反应。在这些反应速率还可以对与肿瘤代谢17,21,22,25,62。代谢途径总结于图2。 <img alt="图2" src="/files/ftp_upload/54751/54751fig1.jpg" /> 图2:丙酮酸的主要代谢反应的示意图。丙酮酸/乳酸盐转化是通过LDH催化，和丙酮酸/丙氨酸转化是通过ALT催化。丙酮酸不可逆地转化为乙酰-CoA和CO 2通过PDH，和CO 2与碳酸氢盐80 pH依赖平衡。 <a href="http://ecsource.jove.com/files/ftp_upload/54751/54751fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 超极化[1- 13 C]丙酮酸及其代谢物的检测已经在大鼠先前证实他本领域37,63 - 65，肝脏66，肌肉和肾62,67。一项研究证明了在正常和禁食大鼠肝66之间的乳酸对丙氨酸的比例显著差异，表现在肝癌68,69高度升高和超极化[1- 13 C]乳酸水平。有证据表明，肿瘤分级可以在使用超极化[1- 13 C]丙酮酸22小鼠前列腺（TRAMP）的转基因腺癌被识别，与超极化乳酸水平表示与切除的肿瘤的组织学分级的高相关性。由ALT从丙酮酸催化丙氨酸也被建议作为大鼠肝癌23的有用标志物。 测量丙酮酸乳酸代谢通量已用于监测缺血63,65,70和与细胞毒性化疗17,40，靶向药物对治疗的响应&lt;SUP&gt; 24,25,41，或在动物模型中放疗26。它也被用于检测在成胶质细胞瘤和乳腺癌的小鼠模型25中的磷脂酰肌醇3-激酶（PI3K）抑制剂LY294002反应。在脑丙酮酸代谢的变化肿瘤26和前列腺癌24,71也被治疗后观察到的。 前列腺癌 前列腺癌是男性死亡相关的全球72老年男性和的第二大癌症的主要癌症。迄今为止，还没有可靠的，非侵入性的方法可用于前列腺癌73,74的早期诊断和表征，强调新颖代谢成像技术，迫切需要使严格的检测和患者分期。前列腺癌被用作模型以证明溶解的DNP 13 CMRSI在患者合并的可能性第57条 。这项工作是继续在第一期临床试验采用[1- 13 C]丙酮酸和13 CMRSI为前列腺癌的成像和它最近刚刚已经完成（NCT01229618）。 这背后的工作的动机是更详细和更广泛的受众13 CMRSI方法与细胞的临床前设置应用程序来说明。测定[1- 13 C]丙酮酸LDH催化代谢[1- 13 C]乳酸盐在 PC3前列腺癌细胞系的体外 ，我们表明溶解的DNP在体外研究的可能的应用和处理的关键步骤和在实验过程中的挑战。

<table><tbody><tr><td>HyperSense DNP Polariser</td><td>Oxford Instruments</td><td></td><td>3.35 T preclinical DNP hyperpolarizer</td></tr><tr><td>GE/Agilent MR901</td><td>GE Healthcare/Agilent Technologies</td><td></td><td>7 T preclinical MRI scanner, with small bore designed for experiments onrodent</td></tr><tr><td>Spinsolve Carbon</td><td>Magritek</td><td></td><td>1 T NMR spectrometer with permanent magnet</td></tr><tr><td>Deuterium Oxide</td><td>Sigma Aldrich</td><td>7789-20-0</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>7558-79-4</td><td></td></tr><tr><td>Sodium phosphate monbasic</td><td>Sigma Aldrich</td><td>7558-80-7</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma Aldrich</td><td>1310-73-2</td><td></td></tr><tr><td>Disodium edetate</td><td>Sigma Aldrich</td><td>6381-92-6</td><td></td></tr><tr><td>Pyruvic acid - &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;1&lt;/sub&gt;</td><td>Cambridge Isotopes Laboratories</td><td>CLM-8077-1</td><td></td></tr><tr><td>Dotarem (0.5 mmol/L)</td><td>Guerbet</td><td></td><td>gadoterate meglumine&amp;nbsp;&amp;nbsp;</td></tr><tr><td>tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2&amp;ndash;4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063)</td><td>GE Healthcare</td><td></td><td>trityl radical used as a sourse of free electron</td></tr><tr><td>PC3 cell line</td><td>ATCC</td><td>CRL1435</td><td></td></tr><tr><td>F-12K medium&amp;nbsp;</td><td>ATCC</td><td>30-2004</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>SCRR-30-2020</td><td></td></tr><tr><td>Trypsine-EDTA Solution, 1x</td><td>ATCC</td><td>30-2101</td><td></td></tr><tr><td>Sample plastic cup</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Trypan blue</td><td>Bio-Rad</td><td>145-0013-MSDS</td><td></td></tr></tbody></table>

hyperpolarized 13c metabolic magnetic resonance spectroscopy and imaging

在过去的几十年里，肿瘤分期，再分期，治疗反应的监测，以及多种癌症复发的检测新方法已经出现连同18架F-氟（[18 F在国家的最先进的正电子发射断层扫描] -FDG PET）。 13 C核磁共振光谱成像（13 CMRSI）是一种微创成像方法，能够代谢的体内和实时监控。作为与基于13 C核磁共振（NMR）的任何其他方法，它面临低热极化和随后的低信噪比的挑战，因为在13℃和其低天然丰度的相对低的旋磁比生物样品。通过克服这些限制，动态核极化（DNP）与随后的样品溶解最近启用常用NMR和磁共振成像（MRI）系统来测量在各种生物系统的研究中，以及图像关键代谢途径。在13 CMRSI使用一个特别有趣的和有希望的分子是[1- 13 C]丙酮酸盐，其中，在过去的十年里，已被广泛用于体外 ，临床前，以及最近，临床研究调查细胞的能量代谢在癌症和其他疾病。在这篇文章中，我们使用的是3.35牛逼临床DNP超极化勾勒溶解DNP的技术，并展示在体外研究它的用法。类似的协议为超极化可以适用于在体内研究的大部分为好。要做到这一点，我们使用乳酸脱氢酶（LDH）和催化[1- 13 C]丙酮酸使用13 CMRSI [1- 13 C]乳酸盐在前列腺癌细胞系，PC3， 体外代谢反应。

的"微波扫描"的结果在图3中示出。它表明，对于[1- 13 C]丙酮酸样品的最佳微波频率为94.156千兆赫的本地3.35-T的超极化。以下所有超极化实验（N = 14）用100毫瓦的功率这种微波频率进行。微波辐射被施加60至80分钟，导致固态超极化高于90％。结果列于图4中。超极化[13℃]的丙酮酸，用5×10 6（N = 2），10 7（N = 2），2×10 7（N = 1），3×10 ...

Watch this Scientific Journal Video about Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging at JoVE.com

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 ...

hyperpolarized-13c-metabolic-magnetic-resonance-spectroscopy

Research

JoVE Journal

Cancer Research

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

10.3K 次观看.  Technische Universität München. 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. 

视频: Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Hyperpolarized Xenon for NMR and MRI Applications

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 T generate only a small detectable net-magnetization of the sample at room temperature 1. Hence, most NMR and MRI applications rely on the detection of molecules at relative high concentration (e.g., water for imaging of biological tissue) or require excessive acquisition times. This limits our ability to exploit the very useful molecular specificity of NMR signals for many biochemical and medical applications. However, novel approaches have emerged in the past few years: Manipulation of the detected spin species prior to detection inside the NMR/MRI magnet can dramatically increase the magnetization and therefore allows detection of molecules at much lower concentration 2.
Here, we present a method for polarization of a xenon gas mixture (2-5% Xe, 10% N2, He balance) in a compact setup with a ca. 16000-fold signal enhancement. Modern line-narrowed diode lasers allow efficient polarization 7 and immediate use of gas mixture even if the noble gas is not separated from the other components. The SEOP apparatus is explained and determination of the achieved spin polarization is demonstrated for performance control of the method.
The hyperpolarized gas can be used for void space imaging, including gas flow imaging or diffusion studies at the interfaces with other materials 8,9. Moreover, the Xe NMR signal is extremely sensitive to its molecular environment 6. This enables the option to use it as an NMR/MRI contrast agent when dissolved in aqueous solution with functionalized molecular hosts that temporarily trap the gas 10,11. Direct detection and high-sensitivity indirect detection of such constructs is demonstrated in both spectroscopic and imaging mode.

The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are given.

超极化氙NMR和MRI的应用

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 ...

科研

工程学

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

溶出动态核极化仪器用NMR实时酶反应速率测量

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

化学

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 ...

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 ...

生物工程

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

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 ...

获得高质量的超极化氙 129 磁共振图像

Troubleshooting and Quality Assurance in Hyperpolarized Xenon Magnetic Resonance Imaging: Tools for High-Quality Image Acquisition

Hyperpolarized (HP) xenon magnetic resonance imaging (129Xe MRI) is a recently federal drug administration (FDA)-approved imaging modality that produces high-resolution images of an inhaled breath of xenon gas for investigation of lung function. However, implementing 129Xe MRI is uniquely challenging as it requires specialized hardware and equipment for hyperpolarization, procurement of xenon imaging coils and coil software, development and compilation of multinuclear MR imaging sequences, and reconstruction/analysis of acquired data. Without proper expertise, these tasks can be daunting, and failure to acquire high-quality images can be&#160;frustrating, and expensive. Here, we present some quality control (QC) protocols, troubleshooting practices, and helpful tools for129Xe MRI sites, which may aid in the acquisition of optimized, high-quality data and accurate results. The discussion will begin with an overview of the process for implementing HP 129Xe MRI, including requirements for a hyperpolarizer lab, the combination of 129Xe MRI coil hardware/software, data acquisition and sequence considerations, data structures, k-space and image properties, and measured signal and noise characteristics. Within each of these necessary steps lies opportunities for errors, challenges, and unfavorable occurrences leading to poor image quality or failed imaging, and this presentation aims to address some of the more commonly encountered issues. In particular, identification and characterization of anomalous noise patterns in acquired data are necessary to avoid image artifacts and low-quality images; examples will be given, and mitigation strategies will be discussed. We aim to make the 129Xe MRI implementation process easier for new sites while providing some guidelines and strategies for real-time troubleshooting.

作者聚焦：使用超极化氙 129 MRI 研究肺部疾病 

Here, we present a protocol for obtaining high-quality hyperpolarized xenon-129 magnetic resonance images, covering hardware, software, data acquisition, sequence selection, data management, k-space utilization, and noise analysis.

超极化氙磁共振成像的故障排除和质量保证：用于高质量图像采集的工具

Hyperpolarized (HP) xenon magnetic resonance imaging (129Xe MRI) is a recently federal drug administration (FDA)-approved imaging modality that produces ...

超极化

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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

骨骼肌疾病的定量磁共振成像

测量超极化 [1-¹³C]硫化的旋转-莱迪兹松弛磁场依赖性

超极化氙磁共振成像的故障排除和质量保证：用于高质量图像采集的工具

超极化氙磁共振成像的故障排除和质量保证：用于高质量图像采集的工具

超极化氙磁共振成像的故障排除和质量保证：用于高质量图像采集的工具