10 February 2007 Acceptor spectral bleedthrough correction in spectral FRET imaging microscopy
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The current advances in fluorescence microscopy coupled with the development of new fluorescent probes such as visible fluorescent proteins (VFP) allow Förster (fluorescence) resonance energy transfer (FRET) to be used to study protein-protein interactions in living specimens. For FRET to occur, the donor emission spectrum should overlap the acceptor absorption at least about 30%. This spectral overlap generates donor and acceptor spectral bleedthrough (DSBT and ASBT) in the FRET channel (or acceptor channel) while exciting the double labeled cell with donor excitation wavelength. The spectral imaging microscopy helps to avoid the DSBT into FRET channel by linear unmixing. But the ASBT is the same spectral region of the FRET signal which cannot be removed by linear unmixing. To obtain a quantitative estimation of the C/EBPα protein dimerization in GHFT1-5 living cell nucleus, we need to remove the ASBT from the FRET channel. So, we developed an algorithm that removes the ASBT signal from the spectral FRET (sFRET) image. The E% estimated using the processed spectral FRET (psFRET) algorithm provides excellent comparison with other techniques such as confocal and lifetime FRET microscopy. This psFRET algorithm was also characterized with FRET standards, based on constructs with known-length amino acid linkers.
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Ye Chen, Ammasi Periasamy, "Acceptor spectral bleedthrough correction in spectral FRET imaging microscopy", Proc. SPIE 6442, Multiphoton Microscopy in the Biomedical Sciences VII, 644223 (10 February 2007); doi: 10.1117/12.710899; https://doi.org/10.1117/12.710899

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