For some time now, we have been using a fluorescence resonance energy transfer (FRET)-based strategy to conduct high-resolution studies of phase behavior in ternary lipid-raft membrane mixtures. Our FRET experiments can be carried out on ordinary, polydisperse multilamellar vesicle suspensions, so we are able to prepare our samples according to a procedure that was designed specifically to guard against artifactual phase separation. In some respects (i.e., the number and nature of two-phase regions observed), our phase diagrams are consistent with those in previously published reports. However, in other respects (i.e., overall size of miscibility gaps, phase boundary locations and their dependence on temperature), there are clear differences. Here, we present FRET data taken in dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine/cholesterol (DOPC/DPPC/Chol) mixtures at 25.0, 35.0, and 45.0 degrees C. Comparisons between our results and previously reported phase boundaries suggest that lipid-raft mixtures may be particularly susceptible to demixing effects during sample preparation.

INTRODUCTIONThe phenomenon of Förster resonance energy transfer (FRET) between two fluorescent chromophores is widely employed for a variety of purposes. 1,2 In this form, FRET may perhaps be best known as a "spectroscopic ruler," serving in applications that exploit the famous inverse sixthorder distance dependence 3 of the so-called transfer efficiency, E . This particular FRET metric is the fractional decrease in donor fluorescence due to acceptor quenching, aso the determination of E requires two separate measurements: donor fluorescence in the presence and absence of acceptor. During the last decade, FRET has seen increasing application in studies of biological membranes, both in model systems 4 and living cells. 2 In these studies, membranes are labeled with two populations of membrane-associated fluorophores (i.e., donor and acceptor probes), and the observed FRET signal is interpreted in terms of either membrane phase behavior or specific interactions between membrane components.Although most of these biomembrane FRET studies have been based on measurements of E , others have chosen to use an alternative metric: donor-excited acceptor fluorescence, of E ; and (iii) whereas measurements of E are sensitive to variations in acceptor concentration only:measurements are sensitive to variations in both probe concentrations:In order to provide for the interpretation of experimental results, freely-diffusing probe studies must resort to a theoretical framework in order to relate variations in the FRET metric to variations in probe distributions. Whereas a common Eχ -using six different combinations of FRET probes and membrane environments. Of course, analyses based on the S-V model are normally applied only to experiments involving collisional quenching. But dilute acceptor concentration is one condition under which Forster kinetics are known to approach the Stern-Volmer limit, b9 so under these circumstances it is also reasonable to employ an S-V model to describe FRET.Our original goal was simply to evaluate the useful limits of an S-V expression forso that we could use it in our FRET-based studies of membrane phase behavior. 10 However, over the course of our research we have discovered that S-V expressions can safely be used to describe both FRET metrics within acceptor-concentration ranges that are conveniently defined by an easily measured parameter: the Stern-Volmer quenching constant. Moreover, we have seen that S-V predictions can even work well up to remarkably b The other condition being "statistical mixing" of excited-state donors and acceptors due to rapid diffusion or excitation migration.[ F has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under diluteprobe conditions. We have also discovered a dilute-regime correspondence between our Stern-Vol...