Tear Down the Fluorescent Curtain: A New Fluorescence Suppression Method for Raman Microspectroscopic Analyses
Tear Down the Fluorescent Curtain: A New Fluorescence Suppression Method for Raman Microspectroscopic Analyses
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DOI：
10.1038/s41598-019-52321-3
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发表时间：
2019-10
影响因子：
4.6
通讯作者：
E. Yakubovskaya;Tatiana Zaliznyak;Joaquín Martínez Martínez-Joaquín-Martínez-Martínez-47930098;G. Taylor
中科院分区：
文献类型：
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作者：
E. Yakubovskaya;Tatiana Zaliznyak;Joaquín Martínez Martínez-Joaquín-Martínez-Martínez-47930098;G. Taylor
在过去的十年中，已发表的拉曼显微光谱应用的近指数增长见证了这项技术的优势和多功能性。然而，激光诱导荧光往往严重阻碍其应用于生物样品。在这里，我们报告了一种新的方法，用于几乎完全消除高度着色的生物标本（例如，微藻），使得能够通过拉曼显微光谱进行询问。我们简单的化学光漂白方法结合了温和的过氧化氢氧化和整个样品的宽光谱可见光照射。这种治疗允许观察细胞内的大分子池，同位素示踪剂，甚至病毒传播的细胞内分布以前不服从拉曼显微光谱检查。我们的方法表明，共焦拉曼显微光谱成为一个不可或缺的工具，以获得高荧光生物样品的化学组成的空间分辨数据，从单个细胞到环境样品的潜力。
The near exponential proliferation of published Raman microspectroscopic applications over the last decade bears witness to the strengths and versatility of this technology. However, laser-induced fluorescence often severely impedes its application to biological samples. Here we report a new approach for near complete elimination of laser-induced background fluorescence in highly pigmented biological specimens (e.g., microalgae) enabling interrogation by Raman microspectroscopy. Our simple chemiphotobleaching method combines mild hydrogen peroxide oxidation with broad spectrum visible light irradiation of the entire specimen. This treatment permits observing intracellular distributions of macromolecular pools, isotopic tracers, and even viral propagation within cells previously not amenable to Raman microspectroscopic examination. Our approach demonstrates the potential for confocal Raman microspectroscopy becoming an indispensable tool to obtain spatially-resolved data on the chemical composition of highly fluorescent biological samples from individual cells to environmental samples.