同步光活化成像荧光团突破活细胞显微镜中光漂白和光毒性的限制,Analytical Chemistry

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同步光活化成像荧光团突破活细胞显微镜中光漂白和光毒性的限制
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-10-27 , DOI: 10.1021/acs.analchem.3c03064
Hong Wang ₁ , Guangmei Han ₁ , Hesen Tang ₁ , Ruilong Zhang ₁ , Zhengjie Liu ₁ , Yingqiang Sun ₁ , Bianhua Liu ₂ , Junlong Geng ₁ , Zhongping Zhang _{1,

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Affiliation

荧光显微镜是细胞生物学和许多其他领域研究中最重要的工具之一，但与荧光团相关的两个基本问题，光漂白和光毒性仍然限制了其在活细胞的长期强照明成像中的应用。在这里，我们报告了荧光团工程化学的新概念，同步光激活成像（SPI）荧光团，通过单一光源激活和激发荧光团，从而避免激活光和激发光之间的重复切换。化学重建的非发射荧光团可以被光解，从而在成像过程中不断补充标准荧光显微镜可检测到的“亮态”探针，从而绕过光漂白屏障，大大延长成像时间。同样重要的是，由于光活化基团清除活性氧 (ROS) 以及缓慢释放“亮态”探针以最大限度地减少 ROS 的产生，SPI 荧光团可显着降低光细胞毒性。使用SPI荧光团，在活细胞中实现了强照明（50 MW/cm ²）下亚细胞细胞器的延时共焦（>16小时）和超分辨率（>3小时）成像。

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Synchronous Photoactivation-Imaging Fluorophores Break Limitations of Photobleaching and Phototoxicity in Live-cell Microscopy

Fluorescence microscopy is one of the most important tools in the studies of cell biology and many other fields, but two fundamental issues, photobleaching and phototoxicity, associated with the fluorophores have still limited its use for long-term and strong-illumination imaging of live cells. Here, we report a new concept of fluorophore engineering chemistry, synchronous photoactivation-imaging (SPI) fluorophores, activating and exciting fluorophores by a single light source to thus avoid the repeated switches between activation and excitation lights. The chemically reconstructed, nonemissive fluorophores can be photolyzed to allow continuous replenishing of “bright-state” probes detectable by standard fluorescent microscopes in the imaging process so as to bypass the photobleaching barrier to greatly extend the imaging period. Equally importantly, SPI fluorophores substantially reduce photocytotoxicity due to the scavenging of reactive oxygen species (ROS) by a photoactivable group and the slow release of “bright-state” probes to minimize ROS generation. Using SPI fluorophores, the time-lapsed confocal (>16 h) and super-resolution (>3 h) imaging of subcellular organelles under intensive illumination (50 MW/cm²) were achieved in live cells.

更新日期：2023-10-27

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