Optical section structured illumination‐based Förster resonance energy transfer imaging

Abstract

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS‐SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3‐cube FRET imaging in OS‐SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS‐SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS‐SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS‐SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were EnormalC32normalVOSF=0.32±0.5em0.02,0.5emEnormalC17normalVOSF=0.38±0.02, and EnormalC5normalVOSF=0.45±0.03, and the measured acceptor‐to‐donor concentration ratios (Rc) were RnormalC32normalVOSF=1.07±0.03, RnormalC17normalVOSF=1.09±0.03, and RnormalC5normalVOSF=1.02±0.04, consistent with the reported values. Collectively, our data demonstrates that OS‐SIM can be integrated into FRET microscopy to build an OS‐SIM‐FRET with confocal microscopy‐like resolution.

Principles of Resonance Energy Transfer

“…imaged biomolecular interactions in the membrane‐associated cytoskeleton of a neuron with high optical resolution (Szalai et al., 2021). Although Structured Illumination Microscopy (SIM) improves optical resolution by only a factor of two, recent application of SIM with FRET made it possible to determine FRET in live HeLa cells with reasonable optical resolution (Liu et al., 2022). Improving SIM could provide an attractive and simple alternative approach to enhance the resolution of FRET images.…”

“…FRET in live HeLa cells with reasonable optical resolution (Liu et al, 2022). Improving SIM could provide an attractive and simple alternative approach to enhance the resolution of FRET images.…”

A mini review of recent development of flow cytometry in China

“…Some other attractive quantitative cell analysis methods have been reported in nanotechnology. Chen et al [16] showed the use of Forster Resonance Energy Transfer (FRET) imaging with optical section structure illumination microscopy for the analysis of living cells. With a home‐built stochastic optical reconstruction microscopy (STORM), Qu et al [17] explored the nanoliposome fine structures that may aid the development of liposome‐based photodynamic therapy for cancer treatment.…”

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

“…This method, called structured illumination-based super-resolution Förster resonance energy transfer microscopy (SIM-FRET), enables dynamic SR quantitative FRET imaging of living cells. 11,12 SIM-FRET achieves high spatiotemporal resolution and minimal photodamage while remaining compatible with commonly used fluorescent protein FRET pairs for conventional live-cell FRET imaging. [12][13][14] Although SIM-FRET has demonstrated its capability to quantify FRET analysis in living cells, the accuracy of quantitative analysis is still greatly affected by SIM reconstruction artifacts.…”

“…11,12 SIM-FRET achieves high spatiotemporal resolution and minimal photodamage while remaining compatible with commonly used fluorescent protein FRET pairs for conventional live-cell FRET imaging. [12][13][14] Although SIM-FRET has demonstrated its capability to quantify FRET analysis in living cells, the accuracy of quantitative analysis is still greatly affected by SIM reconstruction artifacts. To maintain the fidelity of image intensity, SIM-FRET has conventionally employed linear Wiener reconstruction for SR image reconstruction.…”