Laurdan: Clarifying Photophysics and Advancing the Characterization of Extracellular Vesicles

The environment-sensitive fluorescence emission of Laurdan (6-lauroyl-2-dimethylaminonaphthalene) underlies its widespread application in studies of biomolecular assemblies. The first part of this invited contribution clarifies the relevant photophysical concepts of Laurdan. Based on an overview of the literature on the characterization of extracellular vesicles (EVs) using Laurdan, the second part advances Laurdan's use by introducing time-resolved emission spectra (TRES) for EV characterization. As diagnostic applications require distinguishing between EV subtypes, and given that tetraspanins are major protein constituents of EVs, we characterized Laurdan fluorescence in EVs isolated from various tetraspanin knockout cell lines. To resolve differences between these EVs, we recorded TRES and analyzed them using the standard time-dependent fluorescence shift approach. In addition, we applied spectral phasor analysis to the TRES data-a combination that, as far as we are aware, has not previously been explored. This combined approach offers clear advantages over previously employed steady-state methods, as it can distinguish EVs with different protein profiles and provides comprehensive information on both compositional heterogeneity and lipid phase state. Inspection of the TRES phasor trajectories further suggests that Laurdan is sensitive not only to the lipid matrix but also to nonlipid constituents of EVs, such as membrane-associated proteins.