# Offsetting ROS-Mediated Arrest of Endothelial Fenestration Dynamics Permits Long-Term Optical Super-Resolution Imaging Validated by AFM

## TL;DR

This study shows how to reduce phototoxicity in live-cell imaging to accurately observe dynamic cell structures like fenestrations in liver cells.

## Contribution

The study identifies ROS as a key cause of imaging artifacts and provides a framework to preserve cell dynamics during super-resolution imaging.

## Key findings

- ROS generated during imaging arrests fenestration dynamics in liver sinusoidal endothelial cells.
- Using 3D SR-SIM with NAC and BioTracker preserves fenestration dynamics without altering their number or size.
- AFM confirms that ROS impair fenestration dynamics and reveals nanomechanical changes in cells.

## Abstract

Advances
in cell biology create the demand for developing
methods
capable of resolving the structure and dynamics of subcellular organelles
in living cells, which are beyond the reach of classical microscopy.
Live-cell super-resolution fluorescence imaging provides this capability;
however, in practice, its application is limited by phototoxicity,
which perturbs cellular features and interferes with natural mechanisms
of biological processes, providing a biased interpretation. Liver
Sinusoidal Endothelial Cells (LSECs), with their nanoscale fenestrations
that are physiologically critical and highly dynamic structures in
the native state, represent a particularly demanding system for fluorescence-based
microscopy. Here, we identify that photoactivation-generated reactive
oxygen species (ROS) are the principal cause of fenestration arrest
in fluorescence microscopy. By implementing three-dimensional super-resolution
structured illumination microscopy (3D SR-SIM), we systematically
evaluate a range of fluorophores and ROS scavengers to optimize imaging
conditions. By combining BioTracker staining, carbon dioxide-independent
medium supplemented with N-acetylcysteine (NAC), we preserved fenestration
dynamics without altering the number/size of fenestrations. Complementary
atomic force microscopy (AFM) validated that the combination of light
and dye exposure impairs fenestration dynamics through ROS, in the
absence of antioxidant supplementation. Additionally, AFM provides
insights into the cells’ nanomechanical changes upon illumination.
Our findings confirm the mechanism underlying imaging-induced artifacts
in LSECs observed in the literature and provide a broadly applicable
framework for extending live-cell super-resolution microscopy of living
cells.