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. 2010 May;51(5):1157–1172. doi: 10.1194/jlr.M003244

Fig. 3.

Fig. 3.

FRET between DChol and cholesterol-rich microdomain markers DHE, DiD, and BCθ. A and B: FRET between DChol and DHE in LUVs. A: Emission spectra (325–575 nm) of LUVs containing 5% DHE and increasing amount of DChol. The spectra were recorded at 37°C with 300 nm excitation. B: The energy transfer efficiency E% as a function of increasing DChol (mol%). Open circles, E% was calculated from experimental data by E = 1 − IDA/ID, where IDA and ID were donor DHE fluorescence intensity at 373 nm in the presence and absence of acceptor DChol, respectively; lines, E% was calculated using a mathematical model of random distribution of DChol relative to DHE in LUV bilayer as described in Methods. Solid line, R = 15.5; short dashed line, R = 19.1; long dashed line, R = 13.1. C–H: FRET between DChol and DHE in living cells by multi-photon imaging. Images were taken with multi-photon excitation using a 900 nm laser and a D375/50 (350–400 nm) emission filter for DHE (C, E), BGG22 (410–490 nm) emission filter for DChol (D, F). C: DHE emission when L-cells were incubated with DHE only. D: DChol emission when L-cells were incubated with DChol only. E: DHE emission when L-cells were incubated with both DHE and DChol. F: DChol emission when L-cells were incubated with both DHE and DChol. G: Average fluorescence intensity of whole cells (mean ± SE, n = 16–24). H: Average fluorescence intensity of PM (mean ± SE, n = 16–24). **, P < 0.01 t-test, significantly different from DHE only for DHE emission and significantly different from DChol only for DChol emission. FRET between DChol and DiD (I–K) and BCθ (L–N). The excitation laser 408 nm was chosen to only excite the donor DChol and emission filter 680/32 was used to only detect the emission from acceptor DiD and BCθ. Upper panels show FRET between DChol (donor) and DiD (acceptor). I: Image of cells labeled with donor DChol only. J: Image of cells labeled with acceptor DiD only. K: Cells were labeled with both DChol and DiD. Lower panels show FRET between DChol (donor) and Alexa Fluor 660 BCθ (acceptor). L: DChol only. M: Alexa Fluor 660 BCθ only. N: DChol plus Alexa Fluor 660 BCθ.