Lipids–Fluorophores Interactions Probed by Combined Nonlinear Polarized Microscopy

Studying the structural dynamics of lipid membranes requires methods that can address both microscopic and macroscopic characteristics. Fluorescence imaging is part of the most used techniques to study membrane properties in various systems from artificial membranes to cells: It benefits from a high sensitivity to local properties such as polarity and molecular orientational order, with a high spatial resolution down to the single-molecule level. The influence of embedded fluorescent lipid probes on the lipid membrane molecules is however poorly known and relies most often on molecular dynamics simulations, due to the challenges faced by experimental approaches to address the molecular-scale dimension of this question. In this work we develop an optical microscopy imaging method to probe the effect of fluorophores embedded in the membrane as lipid probes, on their lipid environment, with a lateral resolution of a few hundreds of nanometers. We combine polarized-nonlinear microscopy contrasts that can independently address the lipid probe, by polarized two-photon fluorescence, and the membrane lipids, by polarized coherent Raman scattering. Using trimethylamino derivative 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) as model probes, we show that both probes tend to induce an orientational disorder of their surrounding lipid CH-bonds in 1,2-dipalmitoylphosphatidylcholine (DPPC) lipids environments, while there is no noticeable effect in more disordered 1,2-dioleoyl