Dye lipophilicity and retention in lipid membranes: implications for single-molecule spectroscopy

Fluorescence studies of individual lipid vesicles rely on the proper positioning of probes in the lipid milieu. This is true for both positional tags and chemoselective fluorogenic probes that undergo chemical modification following reaction with an analyte of interest within the lipid environment. The present report describes lipophilicity and localization estimations for a series of BODIPY dyes bearing substituents of varying hydrophobicity. We also studied fluorogenic trap-reporter probes that undergo fluorescence emission enhancement upon trapping of reactive oxygen species (ROS), including lipid peroxyl radicals. We show that caution has to be taken to extrapolate ensemble partition measurements of dyes to the single-molecule regime as a result of the dramatically different lipid concentration prevailing in ensemble versus single-molecule experiments. We show that the mole fraction of dyes that remains embedded in liposomes during a typical single-molecule experiment may be accurately determined from a ratiometric single-particle imaging analysis. We further demonstrate that fluorescence correlation spectroscopy (FCS) provides a very rapid and reliable estimate of the lipophilic nature of a given dye under highly dilute single-molecule-like conditions. Our combined single-particle spectroscopy and FCS experiments suggest that the minimal mole fraction of membrane-associated dyes (x(m)) as determined from FCS experiments is about 0.5 for adequate dye retention during single-molecule imaging in lipid membranes. Our work further highlights the dramatic effect that chemical modifications can have on chemoselective fluorogenic probe localization.