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. 2015 Mar 19;50(3):231–241. doi: 10.3109/10409238.2015.1023252

Figure 4.

Figure 4.

Single vesicle–vesicle lipid-mixing assays with freely diffusing vesicles. (A) Schematic representation of the ALEX method (Choi et al., 2013). Dilute mixtures of DiI labeled t-vesicles (donor) are mixed with DiD labeled v-vesicles (acceptor) and imaged using confocal microscopy with alternating laser excitation. The green area represents the confocal detection volume. (B) Representative data collected on individual vesicles diffusing through the confocal volume. Three data channels are recorded as shown: donor emission when directly excited (Ex: D, Em: D); acceptor emission when directly excited (Ex: A, Em: A); and acceptor emission resulting from FRET when the donor is excited (Ex: D, Em: A). The vesicles can be distinguished by their representative fluorescence signals as: t-vesicles only, emission of donor when directly excited, but no acceptor signal; v-vesicle only, no donor signal and emission of acceptor only when directly excited; docked vesicles, emission of both donor and acceptor when excited with the respective excitation sources but no FRET; and fused vesicles resulting in FRET (emission of both donor and acceptor when excited with the green laser). Reproduced with permission from Choi et al. (2013).