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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Macromolecular-scale resolution in biological fluorescence microscopy
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Macromolecular-scale resolution in biological fluorescence microscopy

机译：生物荧光显微镜中的大分子分辨率

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摘要

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximate to 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.
机译：我们展示了在生物样品中焦平面分辨率为15-20 nm的远场荧光显微镜。通过消除分子三重态激发作为激发光耗尽显微镜中许多染料光漂白的主要来源，已使衍射屏障以下的分辨率提高了10至12倍。与报道的受激发射耗尽照明方案相比，允许在随后的激发-耗尽循环之间放宽三重态的产生可使总荧光信号最多增加30倍。此外，它可以使有效焦斑面积减小至比衍射所给定的有效焦点面积低约140倍。通过降低脉冲激光的重复率或通过提高扫描速度可以有效地防止三重态的建立，从而可以实现三重态的弛豫。免疫荧光成像的这种分辨率可通过使用常规光学系统揭示内体上的纳米级蛋白质模式，神经元中中间丝的点状结构以及哺乳动物细胞中的核蛋白斑点来证明。衍射无限制荧光显微镜的报道性能为解决生命科学中的基本问题开辟了道路。

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