用化探针逐步标记:MINFLUX成像和跟踪的一般方法
Longfang Yao1,2, Dongjuan Si3, Liwen Chen4
1School of Artificial Intelligence Science and Technology, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai 200093, China.
Science advances
|May 21, 2025
概括
我们开发了用于MINFLUX (GLF-MINFLUX) 成像和跟踪的化探针的逐步标签. 这种方法在密集的细胞环境中实现了高精度的活细胞蛋白跟踪和成像.
科学领域:
- 生物物理学的生物物理.
- 细胞生物学 细胞生物学
- 纳米技术纳米技术
背景情况:
- 最小光子流 (MINFLUX) 纳米镜对于纳米级蛋白质研究具有强大作用.
- 现有的MINFLUX方法难以在复杂的细胞环境中同时对活细胞进行成像和跟踪.
研究的目的:
- 使用MINFLUX纳米显微镜开发用于同时成像和活细胞跟踪的通用策略.
- 为了提高纳米级蛋白质分析的精度,获取速度和目标密度.
主要方法:
- 开发了用于MINFLUX (GLF-MINFLUX) 的化探针的渐进标签.
- 使用膜通透,蛋白质诱导的"关/开"切换化染料进行逐步标记,定位和漂白.
- 启用了单个蛋白质的顺序定位和跟踪.
主要成果:
- 实现了连续微管的2.6纳米定位精度,优于传统的MINFLUX.
- 精度提高了1.7倍,采集速度提高了2.2倍,目标密度提高了3倍.
- 启用了线粒体蛋白质的3D定位和微管和微纤维的双通道成像.
- 在~200μs分辨率下以7.8nm精度进行活细胞单蛋白跟踪,揭示了明显的扩散行为.
结论:
- 通过调整探头度,GLF-MINFLUX为纳米级蛋白质研究提供了一种多功能方法.
- 提供分子层面的洞察力,了解活细胞中的蛋白质功能和动态.
- 在密集的细胞环境中显著提升活细胞成像和跟踪能力.
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