High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime

Abstract

Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism.

The ever-expanding role of HIF in tumour and stromal biology

“…One hypothesis is that mitochondrial respiration regulates HIF-α stability by increasing intracellular hypoxia, thereby impairing PHD-mediated HIF-α hydroxylation. Indeed, high resolution imaging indicates that mitochondria reside in regions of lower oxygen, compared to other cellular compartments 28 , and mitochondrial ETC inhibitors interfere with hypoxic stabilization of HIF-α, presumably by preserving intracellular oxygen for PHD-mediated hydroxylation 29 . Furthermore, PGC-1α, a central regulator of mitochondrial biogenesis, stabilizes HIF-α by stimulating mitochondrial oxygen consumption and increasing intracellular hypoxia 30,31 .…”

Mitochondrial Function and Cell Size: An Allometric Relationship

“…First, it predicts that the molecules required for energy production should display a gradient towards the centre of the cell. While more quantitative experiments are needed, current experimental evidence suggests that at least oxygen levels are higher close to cell surface and lower in the middle of cells [58]. Second, the size-dependent decline in mitochondrial metabolism should be more pronounced in conditions where cells are highly dependent 10 on mitochondria for energy production.…”

Computational Investigation of O2Diffusion Through an Intra–molecular Tunnel in AlkB; Influence of Polarization on O2Transport

“…23, 2017; these enzymes require a second co-factor (α-kg) and a substrate (1mA for AlkB) to turn over, and are mainly located in the nuclei (and to a lesser degree in mytochondria) where the oxygen concentration is known to be low. 82 Thus, a high affinity for the co-substrate would be beneficial. Although we are not able to perform ligand diffusion simulations based on long MD calculations using the AMOEBA force field due to computational constraints, our results underscore the importance of the inclusion of polarization in certain biological systems.…”