Ultrafast Simultaneous Raman-Fluorescence Spectroscopy.
Ultrafast Simultaneous Raman-Fluorescence Spectroscopy.
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DOI：
10.1021/acs.analchem.9b03563
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发表时间：
2019-11
影响因子：
7.4
通讯作者：
M. Lindley;K. Hiramatsu;Hayate Nomoto;F. Shibata;T. Takeshita;S. Kawano;K. Goda
中科院分区：
文献类型：
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作者：
M. Lindley;K. Hiramatsu;Hayate Nomoto;F. Shibata;T. Takeshita;S. Kawano;K. Goda
拉曼光谱和荧光光谱在生物分析化学中提供了互补的方法，特别是在微生物检测方面。前者用于以无标记的方式检测脂质、代谢物以及非特异性蛋白质和核酸，而后者用于通过标记或转染研究靶向蛋白质、核酸及其相互作用。尽管它们具有互补性，但由于荧光信号比固有微弱的拉曼信号强几个数量级以上，这些方法很少结合使用。在此我们报道一种多模态光谱仪，它能高速同时进行拉曼光谱和荧光光谱分析。它通过傅里叶变换相干反斯托克斯拉曼散射（FT - CARS）和傅里叶变换双光子激发（FT - TPE）测量得以实现，这些测量由一台飞秒脉冲激光器提供动力，该激光器与一台自制的快速扫描迈克尔逊干涉仪相连，每秒可获取24000个光谱。作为原理验证演示，我们通过测量有机溶剂中的香豆素染料验证了超快荧光拉曼光谱仪。为了展示其在需要快速荧光拉曼光谱的应用中的潜力，我们还展示了在不同培养条件下对雨生红球藻细胞进行荧光拉曼流式细胞术分析，其高通量约为每秒10个事件，以对其代谢应激反应进行大规模单细胞分析。
Raman and fluorescence spectroscopies offer complementary approaches in bioanalytical chemistry, particularly in microbiological assays. The former method is used to detect lipids, metabolites, and nonspecific proteins and nucleic acids in a label-free manner, while the latter is used to investigate targeted proteins, nucleic acids, and their interactions via labeling or transfection. Despite their complementarity, these regimes are seldom used in conjunction due to fluorescent signals overwhelming inherently weak Raman signals by more than several orders of magnitude. Here we report a multimodal spectrometer that simultaneously performs Raman and fluorescence spectroscopies at high speed. It is made possible by Fourier-transform-coherent anti-Stokes Raman scattering (FT-CARS) and Fourier-transform-two-photon excitation (FT-TPE) measurements powered by a femtosecond pulse laser coupled to a homemade rapid-scan Michelson interferometer, operating at 24 000 spectra per second. As a proof-of-principle demonstration, we validate the ultrafast fluoRaman spectrometer by measuring coumarin dyes in organic solvents. To show its potential for applications that require rapid fluoRaman spectroscopy, we also demonstrate fluoRaman flow cytometry of Haematococcus pluvialis cells under varying culture conditions with a high throughput of ∼10 events per second to perform large-scale single-cell analysis of their metabolic stress response.