title: Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.
abstract: The orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment.
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France. Electronic address: patrick.ferrand@fresnel.fr.
authors.forename: Patrick
authors.initials: P
authors.lastname: Ferrand
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Paulina
authors.initials: P
authors.lastname: Gasecka
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Alla
authors.initials: A
authors.lastname: Kress
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Fatma-Zohra
authors.initials: FZ
authors.lastname: Bioud
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Duboisset
authors.affiliation: Aix-Marseille Université, CNRS, Centrale Marseille, Institut Fresnel UMR 7249, Marseille, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Brasselet
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry*; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008956:Models, Chemical; D011726:Pyridinium Compounds / Q000737:chemistry*
chemical_list: C089669:1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine; D005456:Fluorescent Dyes; D008081:Liposomes; D011726:Pyridinium Compounds