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.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.

Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting.

A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous HO to living cells or tissues. However, the addition of HO to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of HO on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids.

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.