Terbium ion as RNA tag for slide-free pathology with deep-ultraviolet excitation fluorescence
Terbium ion as RNA tag for slide-free pathology with deep-ultraviolet excitation fluorescence
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DOI：
10.1038/s41598-019-47353-8
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发表时间：
2019-07-24
影响因子：
4.6
通讯作者：
Takamatsu, Tetsuro
中科院分区：
文献类型：
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作者：
Kumamoto, Yasuaki;Matsumoto, Tatsuya;Takamatsu, Tetsuro
深紫外激发荧光显微镜已使分子成像具有光学切片能力与宽场配置和其实用性的无载玻片病理学已被证明在最近几年。在这里，我们报告的有用性铽离子作为RNA特异性标记探针的无载玻片病理学与深紫外激发荧光。在250-300 nm波长范围内激发时，铽离子进入细胞后发出荧光。在染色前核糖核酸酶分解RNA后，在核仁和细胞质中观察到明亮的荧光，而荧光变弱。还发现，在染色过程中，核质的荧光强度随着温度的增加而增加，这种温度依赖性行为类似于由于熔化而导致的DNA的温度依赖性减色。这些发现表明铽离子对单链核酸的染色比双链核酸更有效。我们进一步将铽离子和DNA特异性染料组合用于双色成像。在所获得的图像中，核仁、核质和细胞质被区分开。我们证明了有用的双色成像快速诊断手术标本显示光学切片的未切片组织。目前的研究结果可以增强深紫外激发荧光显微镜，从而扩大荧光显微镜在生命科学中的潜力。
Deep-ultraviolet excitation fluorescence microscopy has enabled molecular imaging having an optical sectioning capability with a wide-field configuration and its usefulness for slide-free pathology has been shown in recent years. Here, we report usefulness of terbium ions as RNA-specific labeling probes for slide-free pathology with deep-ultraviolet excitation fluorescence. On excitation in the wavelength range of 250-300 nm, terbium ions emitted fluorescence after entering cells. Bright fluorescence was observed at nucleoli and cytoplasm while fluorescence became weak after RNA decomposition by ribonuclease prior to staining. It was also found that the fluorescence intensity at nucleoplasm increased with temperature during staining and that this temperature-dependent behavior resembled temperature-dependent hypochromicity of DNA due to melting. These findings indicated that terbium ions stained single-stranded nucleic acid more efficiently than double-stranded nucleic acid. We further combined terbium ions and DNA-specific dyes for dual-color imaging. In the obtained image, nucleolus, nucleoplasm, and cytoplasm were distinguished. We demonstrated the usefulness of dual-color imaging for rapid diagnosis of surgical specimen by showing optical sectioning of unsliced tissues. The present findings can enhance deep-ultraviolet excitation fluorescence microscopy and consequently expand the potential of fluorescence microscopy in life sciences.