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用封闭的单光子相机对单个分子进行广场光寿命成像
Nathan Ronceray1, Salim Bennani2, Marianna Fanouria Mitsioni2,3
1Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland. nathan.ronceray@epfl.ch.
Light, science & applications
|August 3, 2025
概括
我们开发了一种快速,高通量单分子光终身成像显微镜 (FLIM) 方法,使用时门相机. 这种技术可以同时精确测量数千个分子,进步超分辨率显微镜和测序.
科学领域:
- 生物物理学的生物物理.
- 显微镜的使用方法
- 分子成像学分子成像学
背景情况:
- 光终身成像显微镜 (FLIM) 对于分子歧视和环境探测至关重要.
- 传统的与时间相关的单光子计数 (TCSPC) 提供了精度,但限制了吞吐量.
- 时隔摄像机显示出高通量FLIM潜力,但它们的单分子应用仍未得到充分探索.
研究的目的:
- 通过使用商业定时单光子相机,研究快速准确的单分子FLIM的可行性.
- 为高通量单分子生命周期测量开发一个优化的采集方案.
- 为了证明生物应用的并行和时间测量.
主要方法:
- 使用商用时门单光子摄像头进行FLIM采集.
- 实施了优化的采购计划,以提高吞吐量和精度.
- 应用该方法研究标记的孔形成蛋白和单分子Förster共振能量转移 (smFRET).
主要成果:
- 实现了与TCSPC接近的精度的单分子生命周期测量.
- 使用512 × 512像素摄像头,可以同时对3000多个分子进行成像.
- 证明了蛋白质的并行生命周期测量和5-25Hz的时间smFRET.
结论:
- 快速而准确的单分子FLIM可以通过时间门相机实现.
- 这种方法显著增加了FLIM应用程序的吞吐量.
- 该技术对推进多目标单分子局部化显微镜和生物聚合物测序具有前途.
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