Images of cells expressing CFP coupled directly to
YFP through a 15 amino acid linker (CFP-15aa-YFP) were acquired and analyzed using Wide-field FRET (W-FRET) microscopy. Excitation wavelengths as per Figure 1. Motorized Olympus IX-70 (www.olympusamerica.com) epifluorescent microscope equipped with Hamamatsu Orca-2 CCD camera (www.hamamatsuphotonics.com), and excitation and emission filter wheels (Periasamy and Day, 1999). All the hardwares were driven by the Isee imaging system software. Objective lens 60x W NA 1.4. (a) and (b) Single-label donor (CFP), donor excitation (Ex) in the donor and acceptor channel, respectively. (c) and (d) Single-label acceptor (YFP), donor Ex and acceptor Ex, respectively, in the acceptor channel. (e) and (f) Double-label (CFP/YFP), donor Ex/donor channel and acceptor channel (unprocessed FRET), respectively. (g) Double-label, acceptor Ex/acceptor channels. (h) and (i) Gray level pixel distribution of unprocessed FRET and processed FRET (PFRET), respectively. PFRET image after processing clearly demonstrates removal of SBT when compared to (f). Efficiency: false-colored rendering of energy transfer efficiency.
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