Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

Abstract

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

“…8). In another study, equimolar mixtures of DOPC, cholesterol and chicken egg yolk sphingomyelin, which display liquid ordered/liquid disordered phase coexistence, were addressed by polarimetric two-photon microscopy using, among other probes, TMA-DPH [91]. Similarly, these authors' results point to reduced angular freedom of TMA-DPH for cholesterol-enriched bilayers or domains, thus in agreement with our simulations.…”

A molecular theory for two-photon and three-photon fluorescence polarization

“…Numerous studies have focused on confined, highly ordered materials where chromophores are held in crystalline structures, [6][7][8] or samples, such as cell membranes, molecular films or fiber, where they are less rigidly bound to a physical matrix. 9,10 In such instances, the rotational freedom of the targeted species is commonly restricted, enforcing a degree of orientational order relative to the external structure. Whereas polarization-derived information is often restricted to two spatial dimensions, the determination of three-dimensional orientation can also be explored.…”

Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane

“…When the probe alignment in the bilayer is approximately known from well-oriented model systems, as GUVs or PM blebs, as shown here for BChol and B-P-Chol, one can use the method also to study the orientation of the membrane topography relative to the incident electromagnetic field. Separating both effects will be highly interesting but requires further work [131,132].…”