LAUSR

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.