# Revised Diffusion Law Permits Quantitative Nanoscale Characterization of Membrane Organization

## TL;DR

This paper introduces a new method to quantitatively analyze nanoscale membrane domains using STED-FCS, enabling the measurement of key parameters like domain size and diffusion rates.

## Contribution

A revised diffusion law approach allows extraction of five key parameters from STED-FCS data, enabling quantitative nanoscale membrane characterization.

## Key findings

- The method successfully validated the Saffman-Delbrück assumption for self-diffusion in ganglioside nanodomains.
- Quantitative diffusion parameters were extracted from both PtK2 cell membranes and giant plasma membrane vesicles.
- A comprehensive framework for molecular diffusion modes in biological membranes was established.

## Abstract

The formation of
functional nanoscopic domains is an
inherent property
of plasma membranes. Stimulated emission depletion combined with fluorescence
correlation spectroscopy (STED-FCS) has been previously used to identify
such domains; however, the information obtained by STED-FCS has been
limited to the presence of such domains while crucial parameters have
not been accessible, such as size (R
d),
the fraction of occupied membrane surface (f), in-membrane
lipid diffusion inside (D
in) and outside
(D
out) the nanodomains as well as their
self-diffusion (D
d). Here, we introduce
a quantitative approach based on a revised interpretation of the diffusion
law. By analyzing experimentally recorded STED-FCS diffusion law plots
using a comprehensive library of simulated diffusion law plots, we
extract these five parameters from STED-FCS data. That approach is
verified on ganglioside nanodomains in giant unilamellar vesicles,
validating the Saffman-Delbrück assumption for D
d. STED-FCS data in both plasma membranes of living PtK2
cells and giant plasma membrane vesicles are examined, and a quantitative
framework for molecular diffusion modes in biological membranes is
presented.