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Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H
摘要： Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens.
关键词： Fluorescence Lifetime Imaging Microscopy (FLIM),single-cell analysis,NADPH/NADH ratio,NAD(P)H,redox,FAD,fluorescence lifetime redox ratio (FLIRR),NAD(P)H-a2%
更新于2025-11-21 11:24:58
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In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing
摘要： Chronic wounds are difficult to diagnose and characterize due to a lack of quantitative biomarkers. Label-free multiphoton microscopy has emerged as a useful imaging modality capable of quantifying changes in cellular metabolism using an optical redox ratio of FAD/(NADH+FAD) autofluorescence. However, the utility of an optical redox ratio for long-term in vivo monitoring of tissue metabolism has not been robustly evaluated. In this study, we demonstrate how multiphoton microscopy can be used to monitor changes in the metabolism of individual full-thickness skin wounds in vivo. 3D optical redox ratio maps and NADH fluorescence lifetime images identify differences between diabetic and control mice during the re-epithelialization of wounds. These metabolic changes are associated with a transient increase in keratinocyte proliferation at the wound edge. Our study demonstrates that high-resolution, non-invasive autofluorescence imaging can be performed in vivo and that optical redox ratios can serve as quantitative optical biomarkers of impaired wound healing.
关键词： metabolism,optical redox ratio,autofluorescence,multiphoton microscopy,in vivo imaging,diabetes,FAD,NADH,wound healing
更新于2025-09-23 15:23:52
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A novel bioreactor for combined magnetic resonance spectroscopy and optical imaging of metabolism in 3D cell cultures
摘要： Purpose: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized 13C‐pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures. Methods: The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI‐compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three‐dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts. Results: Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized 13C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose‐deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ1 (free NADH) showed no significant change, while τ2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH. Conclusion: A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and 13C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied.
关键词： multimodal,optical imaging,bioreactor,magnetic resonance spectroscopy (MRS),nicotinamide adenine dinucleotide (NADH),metabolism,fluorescence lifetime imaging (FLIM),lactate production
更新于2025-09-23 15:22:29
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Photoregeneration of Biomimetic Nicotinamide Adenine Dinucleotide Analogues via a Dye-Sensitized Approach
摘要： Two-step photochemical reduction of an acridinium-based cation 2O+ to the corresponding anion 2O? was investigated using a dye-sensitized approach involving 2O+?COOH attached to the surface of a wide-bandgap semiconductor, p-NiO. The cation 2O+ and corresponding one-electron reduced radical form, 2O? were synthesized and characterized using steady-state UV/vis and electron paramagnectic resonance spectroscopy. The thermodynamics for the photoinduced hole injection from 2O+ and 2O? were evaluated and found to be favorable. Subsequent femtosecond transient absorption spectroscopy was utilized to evaluate the photoinduced hole injection into NiO, starting from 2O+?COOH/NiO and 2O??COOH/NiO samples. The excitation of 2O+?COOH at 620 nm initiated fast (2.8 ps) hole injection into NiO. However, 90% of the charge-separated population recombined within ～40 ps, while ～10% of the charge-separated population exhibited lifetimes longer than the time scale of our instrument (1.6 ns). In the case of 2O??COOH/NiO, the light absorption occurs predominantly by NiO (2O??COOH absorbs at 310 nm) and is associated with the electron transfer from the conduction band of NiO to the radical. The charge-separated state in this case appears to be long-lived, based on the slow (ns) growth of the trapped carriers formed on the NiO surface. The results of this work indicate that the photochemical reduction of 2O+ to the corresponding hydride form (2OH) can be achieved, opening the possibility of using such a dye-sensitized approach for regeneration of nicotinamide adenine dinucleotide analogues in enzymatic and chemical catalysis.
关键词： multiple charge accumulation,stable radical,NADH,photochemistry,NiO,dye sensitization
更新于2025-09-23 15:21:21
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Self-Assembled Protein/Carbon Nitride/Sulfur Hydrogel Photocatalyst For Highly Selective Solar Chemical Production
摘要： Artificial photosynthesis process is a nature inspired process that can convert solar energy to important value added chemicals. Here, we report the synthesis of a self-assembled protein and carbon nitride/sulfur hydrogel (Fmoc-D-PheA/g-C3N4/S) as a visible light active hydrogel photocatalyst for 86.78% reduced nicotinamide adenine dinucleotide (NADH) regeneration and 90.0% L-Glutamate production under solar light irradiation. The present work represents a new benchmark example for NADH regeneration and solar chemical production.
关键词： NADH regeneration,hydrogel photocatalyst,Carbon nitride
更新于2025-09-19 17:13:59