# Laurdan Adopts Distinct, Phase-Specific Orientations in Lipid Membranes

## TL;DR

This study experimentally confirms that Laurdan adopts different orientations in lipid membranes depending on the membrane phase, affecting its fluorescence.

## Contribution

First experimental validation of Laurdan's phase-specific orientation in lipid bilayers using polarized excitation.

## Key findings

- Laurdan aligns more parallel to the membrane normal in the liquid-ordered phase.
- In the liquid-disordered phase, Laurdan adopts a more planar orientation.
- Emission spectra show deviations at longer wavelengths, especially in the liquid-disordered phase.

## Abstract

For over 40 years, Laurdan has been widely used as a
universal
fluorescent probe for the study of lipid membranes. However, recent
molecular dynamics simulations have uncovered previously unknown properties
of Laurdan, revealing that it can adopt distinct conformations within
the lipid bilayer, thereby influencing its molecular orientation.
Despite these findings, experimental and quantitative validation has
been lacking. Here, we present the first experimental study of the
orientation of Laurdan in a phase-separated supported lipid bilayer,
directly linking its spatial orientation to its emission spectra in
liquid-ordered (Lo) and liquid-disordered (Ld) phases. Using azimuthally and radially polarized excitation beams,
we show that in the Lo phase, Laurdan molecules align more
parallel to the membrane normal, whereas in the Ld phase,
they adopt a more planar orientation within the membrane. Interestingly,
the emission spectra for both excitation modes converge at shorter
wavelengths, but show deviations at longer wavelengths, particularly
in the Ld phase. By refining our understanding of the behavior
of Laurdan in lipid membranes, this study underlines the critical
role of the molecular orientation of the dye in fluorescence-based
membrane studies and highlights the need for orientation-sensitive
analysis in biophysical investigations.