Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy
Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy
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DOI：
10.1021/ac400266a
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发表时间：
2013-05-21
影响因子：
7.4
通讯作者：
Moger, Julian
中科院分区：
文献类型：
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作者：
Mansfield, Jessica C.;Littlejohn, George R.;Moger, Julian
不断增长的世界人口对农业和农化工业提出了越来越高的要求，以提高农业产量。这只能通过投资基础植物和农用化学品研究以及开发改进的分析工具来支持这些领域的研究才能实现。目前缺乏在细胞尺度上对植物组织进行非侵入性结构和化学分析的分析工具。相干反斯托克斯拉曼散射(CARS)和受激拉曼散射(SRS)显微镜等成像技术提供了基于振动光谱的无标记化学特定图像对比度。在过去的十年里，这些技术已经被证明为广泛的生物医学研究应用提供了明显的优势。固有的振动对比度提供了无标记的定量功能分析，它不受光漂白的影响，它允许以亚微米的空间分辨率近实时地进行3D成像。然而，由于目前的检测方案对来自色素(如叶绿素)的光吸收和荧光的敏感性，植物科学和农业化学研究界未能从这些技术中受益，它们在植物研究中的应用几乎仍未被探索。在本文中，我们探讨了在CARS和SRS显微镜下对叶绿素荧光和吸收的影响。我们证明，对于后者，可以使用相敏检测来将振动信号从(电子)吸收过程中分离出来。最后，我们通过对植物细胞壁成分、表层蜡质的原位化学分析以及农用化学制剂在叶表面的沉积来展示SRS在植物中的一系列应用的潜力。
The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.