College of Biophotonics & MOE Lab. of Laser Life Science, South China Normal University, Guangzhou, China.
J Microsc. 2019 Apr;274(1):45-54. doi: 10.1111/jmi.12783. Epub 2019 Feb 1.
Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET imaging) is a popular fluorescence intensity-based FRET quantification method. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. E-FRET imaging was performed on the microscope for living cells expressing different FRET tandem constructs. Dynamical E-FRET imaging on the microscope for live cells coexpressing CFP-Bax and YFP-Bax treated by staurosporine (STS) revealed three Bax redistribution stages: Bax translocation from cytosol to mitochondria within 10 min, membrane insertion with conformational change on mitochondrial membrane within about 30 min, and subsequent oligomerisation within about 10 min. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell. LAY DESCRIPTION: Acceptor-sensitised 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed as E-FRET) is a popular fluorescence intensity-based FRET quantification methods. E-FRET measurements are currently performed manually, and a complete FRET measurement takes about 12 s. E-FRET measurement necessitates not only a skilled operator and specialised equipment but also expertise in the interpretation of FRET signals, a considerable challenge in the application of FRET technology in living cells. Furthermore, manual E-FRET microscope is hard to perform dynamical quantitative FRET measurement, the ever-increasing applications in mapping the biochemical signal transduction within cells. Here, an automated E-FRET microscope with user-friendly interfaces was set up for dynamical online quantitative live-cell FRET imaging. This microscope reduces the time of a quantitative E-FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E-FRET imaging of the cells can be performed automatically by clicking 'Capture' button on interfaces. Because of excellent user-friendly interface and stability, the automated E-FRET microscope is a convenient tool for quantitative FRET imaging of living cell.
受体敏化 3 立方体荧光共振能量转移（FRET）成像（也称为 E-FRET 成像）是一种流行的基于荧光强度的 FRET 定量方法。这里，建立了一个具有用户友好界面的自动化 E-FRET 显微镜，用于动态在线定量活细胞 FRET 成像。该显微镜将定量 E-FRET 成像的时间从 12 秒缩短至 3 秒。在定位细胞后，只需点击界面上的“Capture”按钮，即可自动完成显微镜校准和细胞 E-FRET 成像。该显微镜用于表达不同 FRET 串联结构的活细胞的 FRET 成像。用该显微镜对共表达 CFP-Bax 和 YFP-Bax 的活细胞进行动态 E-FRET 成像，并用星形孢菌素（STS）处理，揭示了 Bax 重分布的三个阶段：Bax 在 10 分钟内从细胞质转位到线粒体，约 30 分钟内在线粒体膜上发生构象变化的膜插入，以及随后的大约 10 分钟内寡聚化。由于具有出色的用户友好界面和稳定性，自动化 E-FRET 显微镜是定量活细胞 FRET 成像的便捷工具。
