We demonstrate a novel bio‐spectroscopic technique, “simultaneous Raman/GFP microspectroscopy”. It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti‐Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two‐component multivariate curve resolution with alternate least squares (MCR‐ALS) analysis in the anti‐Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR‐ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm⁻¹, previously reported as “Raman spectroscopic signature of life of yeast.”

中文翻译：

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细胞器特异性同时拉曼/绿色荧光蛋白显微光谱用于活细胞理化研究。

我们展示了一种新颖的生物光谱技术，“同时拉曼/ GFP显微光谱”。它可以使活细胞的细胞器特异性拉曼光谱。裂殖酵母，裂殖酵母，其线粒体被绿色荧光蛋白（GFP）标记，被用作测试模型系统。拉曼激发激光和GFP激发光同时照射样品酵母细胞。在反斯托克斯区监视GFP信号，该区的拉曼散射干扰很小。值得注意的是，从19个活酵母细胞的不同点测得的13568拉曼光谱根据其GFP荧光强度进行了分类，并在反抗中使用了两分量多元曲线分辨率和交替最小二乘（MCR‐ALS）分析斯托克斯地区。通过这种分类，我们可以知道拉曼光谱是否来自线粒体。线粒体特有的拉曼光谱是通过MCR-ALS分析在1389年强烈GFP阳性光谱的斯托克斯区获得的。已获得两个线粒体特定的拉曼光谱。第一个是蛋白质拉曼带占主导地位，第二个是脂质拉曼带占主导地位，这与已知的线粒体分子组成是一致的。此外，第二个光谱显示在1602 cm处有很强的麦角固醇带^-1，先前报道为“酵母寿命的拉曼光谱特征”。