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Widely accessible method for superresolution fluorescence imaging of living systems

机译：生命系统超分辨率荧光成像的广泛使用的方法

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摘要

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.
机译：超分辨率荧光显微镜克服了衍射分辨率的障碍，使生活中的分子错综复杂的细节得以显露。但是，许多当前的超分辨率技术仍然面临局限性，其实现通常与陡峭的学习曲线相关。基于图案照明的超分辨率技术[例如，激发发射损耗（STED），可逆光学线性荧光跃迁（RESOLFT）和饱和结构化照明显微镜（SSIM）]需要专用设备，而基于单分子的方法[例如，随机光学重建显微镜（STORM），光活化定位显微镜（PALM）和荧光-PALM（F-PALM）]涉及重复的单分子定位，这需要它自己的专业知识，并且在时间上也有要求。在这里，我们提出了一种超分辨率荧光成像方法，即光致变色随机光学波动成像（pcSOFI）。在这种方法中，以适当的波长照射可逆的光开关荧光蛋白会产生强大的单分子强度波动，如前所述，可以通过对每个像素随时间变化的统计分析从中提取超分辨率图片。 SOFI。这种方法使用了现成的可遗传编码的设备，并且可以简单，快速地采集数据，能够提供两到三倍的空间分辨率，显着的背景抑制，显着改善的对比度以及有利的活细胞时间分辨率。 。此外，3D成像和多色成像都可以轻松实现。由于其易用性和高性能，我们预计pcSOFI将被证明是一种用于超分辨率成像的有吸引力的方法。

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  • 作者

    Peter Dedecker; Gary C. H. Mo; Thomas Dertinger; Jin Zhang;

  • 作者单位

    Department of Pharmacology and Molecular Sciences,The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Department of Chemistry, University of Leuven, Celestijnenlaan 200F, Heverlee, Belgium;

    Department of Pharmacology and Molecular Sciences,The Johns Hopkins University School of Medicine, Baltimore, MD 21205;

    SOFAST, 10999 Berlin, Germany;

    Department of Pharmacology and Molecular Sciences,The Johns Hopkins University School of Medicine, Baltimore, MD 21205 The Solomon H. Snyder Department of Neuroscience,The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    subdiffraction-limit; two-color imaging; membrane rafts;

    机译：亚衍射极限两色成像;膜筏;

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