Automated E‐FRET microscope for dynamical live‐cell FRET imaging

Abstract

Summary 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.

High-fidelity SIM reconstruction-based super-resolution quantitative FRET imaging

“…The background of the reconstructed three-channel SR-FRET image is removed based on the average intensity of the background region in each image and then designs a binary mask filter 27 . Then, the donor-centric FRET efficiency (ED) and the concentration ratio of total acceptor to donor (RC) are measured by following equations: 28 , 29 ED=FCFC+G·ISIMDD,RC=k·ISIMAAFC/G+ISIMDD,where G is the sensitivity quenching factor and k is the concentration correction factor.…”

Navigating FRET Imaging Techniques for Biosensor Applications

“…Note that other research groups have proposed further alternative calculations involving up to nine images to correct the calculated sensitized emission FRET values (36). Advised additional literature on advanced SE-FRET approaches can be found in Kaminski et al (41) and Broussard et al (51) and papers covering the 'three-cube method' approach to FRET (52)(53)(54).Clearly, SE-FRET offers a wide toolbox for quantification of FRET, ranging from crude to more refined. While the quantitative possibilities and its applicability in confocal and widefield microscopes are direct advantages, the required measuring of (at least) three samples remains a complication.…”

Optical section structured illumination‐based Förster resonance energy transfer imaging

“…where I AApost represents the acceptor fluorescence intensity after selectively photobleaching partial acceptors; I DDpost represents the donor fluorescence intensity after selectively photobleaching partial acceptors [34]. The background of the images used in the FRET calculation has been subtracted just as described previously [35]. For the 3-cube FRET system, we should ensure that the images are not shifted after changing the filter cubes to avoid FRET measurements failure.…”