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Spectral imaging and linear un‐mixing enables improved FRET efficiency with a novel GFP2–YFP FRET pair
Author(s) -
Zimmermann Timo,
Rietdorf Jens,
Girod Andreas,
Georget Virginie,
Pepperkok Rainer
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03508-1
Subject(s) - förster resonance energy transfer , yellow fluorescent protein , fluorescent protein , fluorescence , green fluorescent protein , chemistry , acceptor , biophysics , physics , biology , optics , biochemistry , gene , condensed matter physics
Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un‐mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2–YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.