Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions.

The complex interplay between hydrogen peroxide (HO) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of HO on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for HO and geNOps for NO, allowing simultaneous imaging in single endothelial cells.

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local HO Signals with Targeted HyPer7.

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (HO) in multiple cell locales (nucleus, cytosol, mitochondria) using the HO biosensor HyPer7.