title: Probing the orientational distribution of dyes in membranes through multiphoton microscopy.
abstract: Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt μ and distribution breadth γ, are more informative than an average tilt angle [φ]. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.
authors.affiliation: Department of Chemistry, University of Oxford, Oxford, United Kingdom. james.reeve@chem.ox.ac.uk
authors.forename: James E
authors.initials: JE
authors.lastname: Reeve
authors.forename: Alex D
authors.initials: AD
authors.lastname: Corbett
authors.forename: Igor
authors.initials: I
authors.lastname: Boczarow
authors.forename: Tony
authors.initials: T
authors.lastname: Wilson
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
authors.forename: Harry L
authors.initials: HL
authors.lastname: Anderson
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004396:Coloring Agents / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009821:Oils / Q000737:chemistry; D011726:Pyridinium Compounds / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000378:metabolism; D014867:Water / Q000737:chemistry
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D004396:Coloring Agents; D009821:Oils; D011726:Pyridinium Compounds; D053835:Unilamellar Liposomes; D014867:Water; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine