The new simple, portable and powerful fluorescence lifetime system and the dedicated FRET calculator that comes standard with the new version of EasyLife V software is ideal to determine the distance between two fluorophores in a multi-phase system, such as micelles, vesicles, liposomes, or membranes.
Experiment
As an example, consider TritonX micellar solution in water with perylene added as the donor and Rhodamine B as the acceptor. Perylene is an aromatic hydrocarbon, virtually insoluble in water, will seek a highly hydrophobic environment and will be localized exclusively inside TritonX micelles.
The plot is shown in Figure 1.
Figure 1
Its fluorescence decay (trace D in the plot) is single exponential with the lifetime of 5.7ns. The Rhodamine B is a hydrophilic molecule and will be mostly in the aqueous phase, however some fraction will also diffuse in the micelles. When both D and A are confined to the same micelle, energy transfer may occur. The perylene decay after the addition of Rhodamine B is shown as trace D+A in the plot.
Now with the addition of the acceptor molecules, the decay is double exponential with the short lifetime of 1.69ns (27%) and the long lifetime of 5.74ns (73%), the latter being the same as for the donor alone (trace D).
Results and Conclusion
The following conclusion can now be drawn
- 27% of all micelles containing perylene also contain Rhodamine B acceptor and the FRET inside the micelles is quite efficient, since the donor lifetime was quenched to 1.69 ns.
- For the Rhodamine B molecules, which are in the aqueous phase the average distance is too great to cause FRET with the micelle-embedded perylene.
- To find the average distance between the D and A in a micelle, open the FRET Calculator, enter the lifetimes of D alone (5.7ns), D in the presence of A (1.69 ns) and Ro = 47.5 Å (determined in a separate experiment see the technical note on the FRET Calculator), click Calculate and the distance is 41.1 Å.
- In addition one gets the FRET efficiency (70%) and the rate constant (4.2x108 s-1). It’s that simple!
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