Imaging of molecular oxygen at subcellular resolution in mammalian cell culture: from 2D to 3D models (764.1)

Abstract

Oxygen monitoring is necessary for studying cell metabolism, stem cell development and ROS production, however most of the current methods suffer from low sensitivity and signal drift (electrodes), lack of quantitative context and end‐point nature (hypoxia stains) or inability to probe O2 inside the cell (extracellular probes). Phosphorescence lifetime imaging microscopy (PLIM) allows for quantitative, sensitive and specific detection of O2, by means of phosphorescent indicator dyes. We developed phosphorescent O2 probe (Pt‐Glc) which demonstrates efficient cytosolic staining of different cell lines (2D cultures), and 3D tissue models such as spheroids (primary neurospheres) and live brain slices from embryonic tissue. The PLIM analysis of neurospheres from rat brain cortex with Pt‐Glc confirmed presence of hypoxic niches. We correlated oxygenation map of spheroids with their size, distribution of live cell (cholera toxin, TMRM), proliferating (BrdU) and stem cell markers (nestin), thus performing multiplexed metabolic imaging of live neurosphere. A comparison with pimonidazole, HIF‐a immunostaining and nanoparticle probes was also made. Thus, Pt‐Glc represents valuable tool for studying stem cell niche within ex vivo and in vitro 3D models.Grant Funding Source: Supported by the Science Foundation Ireland.