Specific visualization of nitric oxide in the vasculature with two-photon microscopy using a copper based fluorescent probe.
Specific visualization of nitric oxide in the vasculature with two-photon microscopy using a copper based fluorescent probe.
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DOI：
10.1371/journal.pone.0075331
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
van Zandvoort MA
中科院分区：
文献类型：
--
作者：
Ghosh M;van den Akker NM;Wijnands KA;Poeze M;Weber C;McQuade LE;Pluth MD;Lippard SJ;Post MJ;Molin DG;van Zandvoort MA
为了研究一氧化氮在各种疾病过程中的作用和（亚）细胞一氧化氮（NO）的构成，在活细胞和组织中直接和特异性地检测一氧化氮是一个重要的要求。有几种方法可用于测量NO的氧化产物，但NO本身的检测已被证明具有挑战性。我们使用NO敏感铜基荧光探针（Cu 2FL2E）和双光子激光扫描显微镜（TPLSM）来观察NO的产生。Cu 2FL2E对NO合成具有较高的敏感性和特异性，并且具有较低的细胞毒性。此外，Cu 2FL2E比常规使用的Griess法显示出更高的灵敏度。通过多种触发NO生成的方法，在体外证实了cu2fl2e在人冠状动脉内皮细胞和猪主动脉内皮细胞中的NO特异性。将TPLSM应用于离体小鼠颈动脉和主动脉，证明了探针对内皮细胞和平滑肌细胞NO生成的适用性。检测血流、乙酰胆碱、过氧化氢等多种刺激下no的生成和时间过程，并证实其与血管舒张的相关性。no合成阻断剂L-NAME的存在消除了no特异性荧光和血管舒张。最后，颈动脉预收缩和血管舒张的影响验证了血管的功能特性。利用Cu 2FL2E-TPLSM对血管NO生成进行特异性可视化，为研究血管生物学中NO的时空合成提供了一种前所未有的有效方法。这种方法可以研究一氧化氮和血管（天）功能之间复杂相互作用的途径。
To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.
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