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Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.

Background: Coenzyme Q10 (CoQ10), also known as ubiquinone-10, is an important molecule of the mitochondrial respiratory chain that acts as an electron carrier between complexes I, II, and III and additionally functions as an antioxidant. Due to its bioenergetic properties, CoQ10 is of high interest for therapeutic and cosmetic use. This study aims to characterize the metabolic impact of CoQ10 on primary human dermal fibroblasts (HDF) using fluorescence lifetime imaging microscopy (FLIM) and electron microscopy. Methods: FLIM of nicotinamide adenine dinucleotide (NADH) is a robust method to characterize cellular energy metabolism that also provides spatial information. Electron microscopy offers a way to characterize the ultrastructure of mitochondria and reveal features not visible in FLIM. Results: We reported a shift towards longer lifetimes of NADH in primary fibroblasts from ten different donors upon treatment with CoQ10, which indicates the stimulation of oxidative phosphorylation. This is confirmed by phasor-based metabolic pattern segmentation, which showed localization of longer NADH lifetimes in CoQ10-treated cells, indicating activated mitochondria in the cytoplasm. In addition, a complementary investigation of the mitochondrial ultrastructure using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography showed a reduction in stress granules in CoQ10-treated cells. Conclusions: Together, FLIM and electron microscopy (EM) imaging strongly imply that CoQ10 stimulates cellular energy metabolism.

Coenzyme Q (CoQ ), also known as ubiquinone-10, is an important molecule of the mitochondrial respiratory chain that acts as an electron carrier between complexes I, II, and III and additionally functions as an antioxidant. Due to its bioenergetic properties, CoQ is of high interest for therapeutic and cosmetic use. This study aims to characterize the metabolic impact of CoQ on primary human dermal fibroblasts (HDF) using fluorescence lifetime imaging microscopy (FLIM) and electron microscopy. FLIM of nicotinamide adenine dinucleotide (NADH) is a robust method to characterize cellular energy metabolism that also provides spatial information. Electron microscopy offers a way to characterize the ultrastructure of mitochondria and reveal features not visible in FLIM. We reported a shift towards longer lifetimes of NADH in primary fibroblasts from ten different donors upon treatment with CoQ , which indicates the stimulation of oxidative phosphorylation. This is confirmed by phasor-based metabolic pattern segmentation, which showed localization of longer NADH lifetimes in CoQ -treated cells, indicating activated mitochondria in the cytoplasm. In addition, a complementary investigation of the mitochondrial ultrastructure using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography showed a reduction in stress granules in CoQ -treated cells. Together, FLIM and electron microscopy (EM) imaging strongly imply that CoQ stimulates cellular energy metabolism.

Background Hyperbaric oxygen therapy (HBOT) has been proposed as a treatment for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), but evidence remains limited. This study evaluated its clinical effectiveness and feasibility, as well as associated functional brain changes. Methods Thirty patients with ME/CFS (mean age 42.3 ± 11.7 years; 7 males, 23 females) received 40 HBOT sessions. Clinical outcomes were assessed at baseline, during treatment, and four weeks post-treatment. The primary outcome was change in the physical functioning subscale of the Short Form-36 Health Survey (SF-36 PF). Secondary outcomes included severity of core symptoms assessed via questionnaires, exercise capacity, handgrip strength, cognitive performance, orthostatic intolerance, and brain magnetic resonance imaging (MRI; volumetry and functional connectivity [FC]). Thirty age- and sex-matched healthy controls (mean age 42.3 ± 11.3 years; 7 males, 23 females) were included for MRI comparison. Results SF-36 PF significantly improved during HBOT compared with baseline (g = 0.71, p  = 0.006). SF-36 pain ( p  = 0.002, g = 0.79) and Chalder Fatigue Scale also showed clinically meaningful reductions ( p  < 0.001, g = −0.87). Exercise capacity (g = 0.66), muscle strength (g = 0.40), and information processing speed (g = 0.52) improved significantly after treatment (all p  < 0.05). Treatment adherence was high and tolerability was favorable, with no major adverse events reported. Functional MRI analyses revealed increased thalamic FC in ME/CFS patients compared to healthy controls in bilateral sensorimotor ( p  < 0.001, t  = 5.65, FDR-corrected) and visuo-occipital regions ( p  < 0.001, t  = 5.40, FDR-corrected) at baseline. Following HBOT, thalamic hyperconnectivity shifted toward patterns observed in healthy controls. Responders, defined as a ≥ 10 points increase in SF-36 PF, showed greater reductions in thalamic hyperconnectivity than non-responders ( p  < 0.001, t  = −4.34 to −5.18, FDR-corrected). Conclusions HBOT was well tolerated and associated with significant improvements in physical functioning, fatigue, pain, and cognitive performance in ME/CFS. The post-treatment shift in thalamocortical connectivity toward healthy control patterns and its association with clinical response support the hypothesis that functional thalamic dysregulation contributes to ME/CFS pathophysiology and may be modulated by HBOT. This provides a network-level rationale for controlled trials to confirm therapeutic efficacy. Trial registration ClinicalTrials.gov NCT06118138. Registered 01 November 2023 – Retrospectively registered, https://clinicaltrials.gov/study/NCT06118138?cond=ME%2FCFSamp;term=HBOTamp;rank=1 .