Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

中文翻译：

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FLEX：使用工程过氧化物酶进行基因编码酶荧光信号放大

生物分子的荧光标记使其能够在分离过程中进行灵敏检测并确定其亚细胞位置。在这种情况下，基于过氧化物酶的反应被积极用于信号放大。为了利用这一潜力，我们使用 APEX (FLEX) 开发了一种基因可编码的酶荧光信号放大方法。我们合成了一种荧光探针，Jenfluence triazole (JFT1)，它可以在固定条件下有效地放大和限制荧光信号，从而能够基于荧光检测亚细胞局部富电子代谢物。此外，即使在锇处理和聚合物嵌入的条件下，JFT1 也表现出稳定的荧光信号，这支持了使用 APEX 的相关光学和电子显微镜 (CLEM) 的发现。使用针对不同细胞器的各种 APEX 缀合感兴趣蛋白 (POI)，我们成功地通过 FLEX 成像可视化它们的定位，同时有效保留细胞器超微结构。 FLEX 提供了对暴露于化学应激源时动态溶酶体-线粒体相互作用的见解。总体而言，FLEX 作为一种灵敏且多功能的系统，在多尺度生物样品中荧光检测 APEX2-POI 方面具有重大前景。