Nanodomain formation in lipid bilayers I: Quantifying the nanoscopic miscibility transition with FRET

We present a robust and easy-to-use methodology for determining the nanoscopic miscibility transition temperature, $\text{nm-}{T}_{mix}$ , of lipid bilayer mixtures from FRET measurements. The method relies on the use of freely diffusing fluorescent donor and acceptor lipids that partition non-uniformly between coexisting phases. When this condition is met, changes in lipid clustering that occur as the sample passes through the transition result in abrupt changes in the spatial distribution of probes and consequently, abrupt changes in the FRET signal. FRET vs. temperature data can then be modeled with a phenomenological piecewise function that describes how the signal changes above and below $\text{nm-}{T}_{mix}$ . Using lattice simulations, we show that the transition between these regimes occurs when the size of lipid clusters surpasses a critical threshold that is approximately equal to twice the Förster distance of the donor/acceptor pair, or about 10 nm. Because other, temperature-dependent factors unrelated to lateral organization-such as changes in lipid molecular area and donor photophysics-can also influence the FRET signal, we also fit the data using a simpler model of uniform mixing. An information theory-based test comparing the fit quality of the uniform and phase separated models provides a straightforward and robust criterion for objectively assessing whether a given sample undergoes a nanoscopic miscibility transition within a temperature range of interest. We highlight the distinction between $\text{nm-}{T}_{mix}$ determined by FRET (or methods with comparable spatial resolution) and the micron-scale transition temperature, $\text{μm-}{T}_{mix}$ , determined from diffraction-limited optical techniques. The analysis software is freely available from an online repository.