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<p>Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (<Emphasis Type="Underline">F</Emphasis>luorescent <Emphasis Type="Underline">L</Emphasis>abeling for <Emphasis Type="Underline">E</Emphasis>xchangeable, <Emphasis Type="Underline">X</Emphasis>-resilient <Emphasis Type="Underline">T</Emphasis>agging in <Emphasis Type="Underline">A</Emphasis>dvanced <Emphasis Type="Underline">G</Emphasis>eneric Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12–18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports extended durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed an innovative protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.</p>

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FLEXTAG: a small and self-renewable protein labeling system for anti-fading multi-color super-resolution imaging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月9日

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  • Han Zhang,
  • Yuan Yao,
  • Xuye Wang,
  • Yuanmin Zheng,
  • Shaoqing Zhang,
  • Yuan Tao,
  • Haopeng Yang,
  • Yinqi Wang,
  • Mengde Liu,
  • Marina Feric,
  • Ruobo Zhou

摘要

Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (Fluorescent Labeling for Exchangeable, X-resilient Tagging in Advanced Generic Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12–18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports extended durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed an innovative protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.