A FLIM Microscopy Based on Acceptor-Detected Förster Resonance Energy Transfer

Abstract

Time-resolved donor-detected Förster resonance energy transfer (trDDFRET) allows the observation of molecular interactions of dye-labeled biomolecules in the ∼10–100 Å region. However, we can observe longer-range interactions when using time-resolved acceptor-detected FRET (trADFRET), since the signal/noise ratio can be improved when observing the acceptor emission. Therefore, we propose a new methodology based on trADFRET to construct a new fluorescence lifetime microscopy (FLIM-trADFRET) technique to observe biological machinery in the range of 100–300 Å in vivo, the last frontier in biomolecular medicine. The integrated trADFRET signal is extracted in such a way that noise is canceled, and more photons are collected, even though trADFRET and trDDFRET have the same rate of transfer. To assess our new methodology, proof of concept was demonstrated with a set of well-defined DNA scaffolds.

Single-molecule acceptor rise time (smART) FRET for nanoscale distance sensitivity

“…In contrast, the acceptor rise time is spectroscopically separated from photophysical heterogeneity, providing a higher signal-to-noise readout for FRET determination. Essentially, the rise time directly isolates the FRET process, which leads to a higher signal-to-noise ratio than indirect methods 25 . Collectively, these advantages are reflected in the improved performance of smART FRET.…”

Off–On Squalene Epoxidase-Specific Fluorescent Probe for Fast Imaging in Living Cells

“…However, formalin used to treat the samples should largely limit its application in living cells . Relatively, a fluorescent probe has been widely used in vitro and vivo with respect to its merits such as noninvasion, high selectivity, and high resolution. − Thereinto, aggregation-induced emission (AIE) active fluorophores exhibit unparalleled advantages and have been successfully used in nonsubstrate fluorescent probes. − Meanwhile, terbinafine is one of the most common inhibitors of SQLE . According to the crystal data (PDB: 6C6N) of the inhibitor/SQLE complex, the key recognition structure of the inhibitor is the hydrophobic side chain .…”