Limitations of Electronic Energy Transfer in the Determination of Lipid Nanodomain Sizes

Abstract

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L(o)) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K(D) = 5 and acceptors K(A) = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L(d)) or L(o) phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.

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“…However, despite advances in far-field optical microscopy, subwavelength lipid domains have never been directly observed, although their sizes are predicted to be <20 nm [19]. FRET is therefore often used to characterize these nano-sized domains [15], because it can detect the nature of nano-domains even if they are transient [20]. The phase diagrams …”

Dynamic label-free imaging of lipid nanodomains

“…Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10)(11)(12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14,15).…”

“…The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14,15). As a result, lipid nanodomain dynamics have not been observed directly even in artificial systems, although recent ensemblebased techniques report lipid heterogeneity on the appropriate length scales (13). In addition to fluorescence-based approaches, ellipsometry and reflection interference contrast microscopy have been used to characterize phase separation in lipid bilayers (16,17), taking advantage of different bilayer thicknesses and refractive indices caused by varying degrees of cholesterol content and lipid packing.…”

Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

“…On the contrary to the previous work [13], D:s and A:s are assumed to reside only at the lipid/water interface. Concerning pores, the donors are assumed to be distributed at the bottom/top part of the pores, while the acceptors are distributed over the entire pore surface ( cf.…”

“…The present paper is a continuation of our previous work [13] and aims at showing how FRET experiments should be performed and the data analyzed in order to achieve the best possible resolution in estimating nanodomain and pore sizes. On the contrary to the previous work [13], D:s and A:s are assumed to reside only at the lipid/water interface.…”