两个FLIM显微镜之间的"激进"差异影响了细胞信号动态的解释
Sravasti Mukherjee1,2, Jeffrey Klarenbeek1, Farid El Oualid3
1Department of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, the Netherlands.
iScience
|July 22, 2024
概括
叶酸在成像介质中的微妙光毒性引起了人工的短暂cAMP信号,与通过对焦时间相关单光子计数 (TCSPC) 观察到的持续反应形成鲜明对比. 这凸显了需要仔细的实验设计和全面的化学披露的需要.
科学领域:
- 细胞生物学 细胞生物学
- 生物物理学的生物物理.
- 药理学 药理学是指药理学的学科.
背景情况:
- 细胞信号的动态对于生物学结果至关重要.
- 光终身成像共振能量转移 (FLIM/FRET) 生物传感器用于研究细胞内信号.
- β1受体-Gαs-cAMP信号轴是细胞通信中的一个关键途径.
研究的目的:
- 使用FLIM/FRET生物传感器研究细胞内信号传输动力学.
- 为了比较使用广场频域FLIM (fdFLIM) 和对焦时间相关单光子计数 (TCSPC) 的cAMP信号动力学.
- 为了确定观察到的cAMP反应动力学的差异的原因.
主要方法:
- 使用了广场频域FLIM (fdFLIM) 和快速对焦时间相关的单光子计数 (TCSPC) 设置.
- 使用FLIM/FRET生物传感器监测β1受体-Gαs-cAMP信号轴.
- 研究了潜在的光诱导效应和介质组件相互作用.
主要成果:
- fdFLIM意外地发现了短暂的cAMP反应,而TCSPC在HeLa和Cos7细胞中检测到持续的反应.
- 没有发现直接的光诱导对cAMP产生或破坏的影响.
- 图像介质中的叶酸,在蓝光激发后,使β1激动剂的降解变得敏感,导致人工信号.
结论:
- 微妙的光毒性,特别是叶酸激发的光毒性,可以显著影响观察到的细胞内信号的动力学.
- 同焦点TCSPC被提倡用于可靠地分析信号响应动力学.
- 科学供应商对化学配方进行全面披露对于实验可重复性至关重要.
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