# High‐Speed Interferometric Scattering Tracking Microscopy of Compartmentalized Lipid Diffusion in Living Cells

## TL;DR

This paper uses high-speed microscopy to study how lipids move in cell membranes, revealing patterns of diffusion and compartmentalization.

## Contribution

The study introduces a high-speed interferometric scattering tracking method to analyze lipid diffusion modes in living cells.

## Key findings

- Lipid diffusion in cell membranes shows transient compartmentalization with an average compartment size of 100–110 nm.
- Diffusion modes include free, confined, transient compartmentalized, and anomalous diffusion, with distinct proportions identified.
- Larger gold nanoparticles affect diffusion rate and confinement strength but not the underlying diffusion modes.

## Abstract

Lateral diffusion measurements have been ‐used to infer information about the nano‐organization of membranes. We employed interferometric scattering (ISCAT) microscopy at an acquisition rate of 2 kHz to revisit the diffusion dynamics of a phospholipid analog on the plasma membrane of Ptk2 cells. The ISCAT trajectory data are analyzed with an unbiased, statistics‐driven pipeline to identify the most likely diffusion mode from a set of plausible diffusion modes. At the ensemble average level, the data are best described as transient compartmentalized diffusion with an average compartment size of 100–110 nm, transient confinement time of 8–10 ms, intracompartmental diffusion coefficient of 0.7–0.9 μm2 s−1, and intercompartmental diffusion coefficient of 0.3–0.4 μm2 s−1. The same analysis applied at the single‐trajectory level identifies a complex variety of diffusion modes with 7–8% free, 13–14% confined, 40% transient compartmentalized, and 40% anomalous diffusion. Measurements with larger (Ø40 nm) as compared to smaller (Ø20 nm) gold nanoparticles are found to influence the diffusion rate and confinement strength, but not the underlying lipid diffusion modes. Using Monte Carlo simulations, these experimental results are explored in the wider context of relevant literature. This analysis paints a unifying picture of lipid diffusion on mammalian cell membranes transcending differences between experimental techniques.

Scheme for the labeling of the cell membrane. The scattering tags, gold nanoparticles of two different diameters (Ø20 nm and Ø40 nm), target the biotinylated lipids inserted in the cellular membrane, owing to their streptavidin coating. The possible, although not certain, effects of cross‐linking are also highlighted (dashed line).© 2025 WILEY‐VCH GmbH