title: Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment.
abstract: Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment.
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada; Department of Physics, University of Toronto, Toronto, Ontario M5S 1A7, Canada.
authors.forename: Zhenfu
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada; Department of Physics, University of Toronto, Toronto, Ontario M5S 1A7, Canada.
authors.forename: Dan
authors.initials: D
authors.lastname: Yomo
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada; Department of Physics, University of Toronto, Toronto, Ontario M5S 1A7, Canada. Electronic address: claudiu.gradinaru@utoronto.ca.
authors.forename: Claudiu
authors.initials: C
authors.lastname: Gradinaru
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008055:Lipids / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
chemical_list: D005456:Fluorescent Dyes; D008055:Lipids; D011506:Proteins
keywords: Adsorption; FCS; Fluorescent dyes; Lipids; Single-molecule spectroscopy