Photodynamic therapy (PDT) that employs the photochemical interaction of light, photosensitizer and oxygen is an established modality for the treatment of cancer. However, dosimetry for PDT is becoming increasingly complex due to the heterogeneous photosensitizer uptake by the tumor, and complicated relationship between the tissue oxygenation ([ ³ O ₂ ]), interstitial light distribution, photosensitizer photobleaching and PDT effect. As a result, experts argue that the failure to realize PDT’s true potential is, at least partly due to the complexity of the dosimetry problem. In this study, we examine the efficacy of singlet oxygen explicit dosimetry (SOED) based on the measurements of the interstitial light fluence rate distribution, changes of [ ³ O ₂ ] and photosensitizer concentration during Photofrin-mediated PDT to predict long-term control rates of radiation-induced fibrosarcoma tumors. We further show how variation in tissue [ ³ O ₂ ] between animals induces variation in the treatment response for the same PDT protocol. PDT was performed with 5 mg kg ⁻¹ Photofrin (a drug-light interval of 24 h), in-air fluence rates ( ϕ _air ) of 50 and 75 mW cm ⁻² and in-air fluences from 225 to 540 J cm ⁻² . The tumor regrowth was tracked for 90 d after the treatment and Kaplan–Meier analyses for local control rate were performed based on a tumor volume ⩽100 mm ³ for the two dosimetry quantities of PDT dose and SOED. Based on the results, SOED allowed for reduced subject variation and improved treatment evaluation as compared to the PDT dose.

Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure (pO₂). This study develops and evaluates a finite-element model of oxygen-consuming mitochondrial bioenergetics using the COMSOL Multiphysics program. The model derives steady-state oxygen (O₂) distributions from Fickian diffusion and Michaelis–Menten consumption kinetics in the mitochondria and cytoplasm. Intrinsic model parameters such as diffusivity and maximum consumption rate were estimated from previously published values for isolated and intact mitochondria. The model was compared with experimental data collected for the intracellular and mitochondrial pO₂ levels in human cervical cancer cells (HeLa) in different respiratory states and under different levels of imposed pO₂. Experimental pO₂ gradients were measured using lifetime imaging of a Förster resonance energy transfer (FRET)-based O₂ sensor, Myoglobin-mCherry, which offers in situ real-time and noninvasive measurements of subcellular pO₂ in living cells. On the basis of these results, the model qualitatively predicted (1) the integrated experimental data from mitochondria under diverse experimental conditions, and (2) the impact of changes in one or more mitochondrial processes on overall bioenergetics.

Mitochondrial endonuclease G (EndoG) contributes to chromosomal degradation when it is released from mitochondria during apoptosis. It is presumed to also have a mitochondrial function because EndoG deficiency causes mitochondrial dysfunction. However, the mechanism by which EndoG regulates mitochondrial function is not known. Fat accumulation in metabolic dysfunction–associated steatotic liver disease (MASLD), which is more common in men, is caused in part by mitochondrial dysfunction. EndoG expression is reduced in MASLD liver, and EndoG deficiency causes MASLD in an obesity-independent manner but only in males. EndoG promotes mitochondrial respiration by resolving mitochondrial tRNA/DNA hybrids formed during mtDNA transcription by recruiting RNA helicase DHX30 to unwind them. EndoG also cleaves off the 3′-end of the H-strand transcript that can prevent mt-tRNA ^Thr precursor cloverleaf-folding, and processing, which increases mt-tRNA ^Thr production and mitochondrial translation. Using fluorescent lifetime imaging microscopy technology to visualize oxygen consumption at the individual mitochondrion level, we found that EndoG deficiency leads to the selective loss of a mitochondrial subpopulation with high-oxygen consumption. This defect was reversed with mt-tRNA ^Thr supplementation. Thus, EndoG promotes mitochondrial respiration by selectively regulating the production of mt-tRNA ^Thr in male mice.

Wiskott–Aldrich syndrome protein family member 3 (WASF3) disrupts mitochondrial respiration and is induced in association with endoplasmic reticulum stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Because cognitive impairment is a hallmark of ME/CFS, we investigated the role of WASF3 in brain metabolism and function using WASF3 transgenic and knockout mouse models. WASF3 disrupted respiratory complex IV and localized to mitochondria-associated membranes, known to modulate cellular processes important for neuronal activity. Overexpression of WASF3 in the hippocampus, a critical region for memory formation, induced a metabolic shift toward glycolysis and promoted a “hypometabolic” state. Increased WASF3 also caused a reduction in ATPIF1, previously shown to facilitate cognitive function, and impaired learning. Conversely, WASF3 deficiency increased ATPIF1 level, enhanced oxidative metabolism, and preserved cognitive function under stress. Together, these findings demonstrate that WASF3 suppresses oxidative metabolism in the brain and contributes to cognitive dysfunction, providing mechanistic insights into the “brain fog” reported in ME/CFS.

As primary sites for oxygen consumption and energy production via oxidative phosphorylation (OXPHOS), mitochondria play a central role in the regulation of bioenergetics and generation of key metabolic intermediates for myogenic cell growth. Common methods to study mitochondria and their metabolism typically rely on population-level analyses, which can mask potential differences in individual cells. In this study, we used various imaging approaches to investigate the interplay between intracellular oxygenation, mitochondrial metabolism and dynamics in a model of myogenic differentiation. Fluorescence imaging of intracellular oxygen revealed that myogenic differentiation is accompanied by progressive shifts in intracellular oxygenation that depend upon and reflect changes in mitochondrial metabolism (i.e., higher oxygen consumption and adenosine triphosphate (ATP) production). By measuring intracellular oxygenation, we showed that mitochondrial metabolism reduces oxygen availability in the cytosol and the nucleus. Real-time redox imaging at the single-cell level further highlighted substantial metabolic heterogeneity and a shift toward OXPHOS as differentiation progressed. Morphological analyses revealed that during myogenic differentiation, mitochondria increase in size while becoming less mobile and overlapping less with microtubules. Overall, this study illustrates the value of combining complementary imaging approaches to provide a comprehensive single-cell perspective on mitochondrial metabolism, remodeling and spatial organization during myogenesis.