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Bioingegneria

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>在体内</em

Published: September 26, 2016
doi:
10.3791/54068
, , , , , , ,
1Department of Chemistry and Biochemistry,University of Denver, 2Magnetic Imaging Group, Applied Physics Division, Physical Measurements Laboratory,National Institute of Standards and Technology, 3Department of Radiology, Geisel School of Medicine,Dartmouth University, 4Department of Biochemistry,West Virginia University, 5Department of Electrical and Computer Engineering,University of Denver, 6Department of Engineering,University of Denver

Summary

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。

Abstract

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。

Introduction

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“>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。

Protocol

1.在250 MHz的快速扫描线圈驱动的安装 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。 确定…

Representative Results

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?…

Discussion

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…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Keywords: Rapid-scan EPR ImagingRSEPRIElectron Paramagnetic ResonanceOxygen ConcentrationPHRedox StatusSignaling MoleculesBiomedical ResearchRicerca sul cancroTumor EnvironmentContinuous Wave EPRRapid-scan Experimental ConditionsResonator BandwidthRapid-scan Coil DriverN-15 PDTBMPOHydrogen PeroxideUV Irradiation
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Biller, J. R., Mitchell, D. G., Tseytlin, M., Elajaili, H., Rinard, G. A., Quine, R. W., Eaton, S. S., Eaton, G. R. Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo. J. Vis. Exp. (115), e54068, doi:10.3791/54068 (2016).
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