Redox Ratio Research Articles

Measuring Metabolic Changes in Cancer Cells Using Two-Photon Fluorescence Lifetime Imaging Microscopy and Machine-Learning Analysis.

Two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) was used to track cellular metabolism with drug treatment of auto-fluorescent coenzymes NAD(P)H and FAD in living cancer cells. Simultaneous excitation at 800 nm of both coenzymes was compared with traditional sequential 740/890 nm plus another alternative of 740/800 nm, before and after adding doxorubicin in an imaging time course. Changes of redox states at single cell resolution were compared by three analysis methods: our recently introduced fluorescence lifetime redox ratio (FLIRR: NAD(P)H-a2%/FAD-a1%), machine-learning (ML) algorithms using principal component (PCA) and non-linear multi-Feature autoencoder (AE) analysis. While all three led to similar biological conclusions (early drug response), the ML models provided statistically the most robust significant results. The advantage of the single 800 nm excitation of both coenzymes for metabolic imaging in above mentioned analysis is demonstrated.

A respiro-fermentative strategy to survive nanoxia in Acidobacterium capsulatum.

Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, the microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum, to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1µM oxygen (3.6 to 0.036 pO2% present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport and catabolism based on COG categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to a NADH/NAD+ redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD+ redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally-relevant soil strains.

Multispectral Imaging of Collagen, NAD(P)H and Flavin Autofluorescence in Mesenchymal Stem Cells Undergoing Trilineage Differentiation.

Understanding the molecular mechanisms of differentiation is important for regenerative medicine and developmental biology. This study aims to characterise the role of the glycolysis/oxidative phosphorylation balance as a driver of mesenchymal stem cell (MSC) differentiation. Cells were maintained in normal conditions or stimulated towards the MSC trilineage cell types over 21 days. Multispectral imaging of cell autofluorescence was applied as a non-invasive methodology to continuously image cultures in situ. Spectral signals for collagen, NAD(P)H, and flavins were unmixed. MSCs cultured under chondrogenic conditions exhibited increased collagen levels relative to controls. Following osteogenic induction, MSCs showed increased collagen levels relative to controls during the earlier stages of culture; however, control cells increased their collagen levels as they became confluent. MSCs cultured under adipogenic conditions exhibited lower levels of collagen than controls. The redox ratio (RR; NAD(P)H/flavins) immediately decreased during chondrogenesis, with this early effect persisting throughout the culture compared to control cells, which appeared to increase their RR, similar to osteogenesis. Adipogenesis resulted in a small increase in RR on day 2 relative to control cells, followed by a persistent decrease. Chondrogenic and adipogenic differentiation favoured oxidative phosphorylation, whereas osteogenesis and MSC overgrowth resulted in a glycolytic metabolism. Following consideration of these findings, as well as the diverse reports in the literature, it is concluded that neither enhanced oxidative phosphorylation nor glycolysis are fundamental to the canonical modes of differentiation, and researchers should avoid interpreting shifts as indicating differentiation.

Metabolic light absorption, scattering, and emission (MetaLASE) microscopy.

Optical imaging of metabolism can provide key information about health and disease progression in cells and tissues; however, current methods have lacked gold-standard information about histological structure. Conversely, histology and virtual histology methods have lacked metabolic contrast. Here, we present metabolic light absorption, scattering, and emission (MetaLASE) microscopy, which rapidly provides a virtual histology and optical metabolic readout simultaneously. Hematoxylin-like nucleic contrast and eosin-like cytoplasmic contrast are obtained using photoacoustic remote sensing and ultraviolet reflectance microscopy, respectively. The same ultraviolet source excites endogenous Nicotinamide adenine dinucleotide (phosphate), flavin adenine dinucleotide, and collagen autofluorescence, providing a map of optical redox ratios to visualize metabolic variations including in areas of invasive carcinoma. Benign chronic inflammation and glands also are seen to exhibit hypermetabolism. MetaLASE microscopy offers promise for future applications in intraoperative margin analysis and in research applications where greater insights into metabolic activity could be correlated with cell and tissue types.

Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy.

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism.

Determination of the Redox Ratio of Iron in a Diamagnetic Matrix Using Low-Field NMR Relaxometry.

Proton nuclear magnetic resonance (NMR) relaxometry is proposed to determine the iron redox ratio of slag samples. It depends on the paramagnetic effect on bulk water relaxation times. Experiments have been carried out to calibrate the relationship between the concentration of Fe3+ and Fe2+ ions and the measured relaxation times on prepared standard samples. It is shown that Fe2+ ions have very different effects on the relaxation times than Fe3+ ions. For the solid slag samples, digestion in an acid was used to produce solutions with a pH of 1.0. Hydrogen peroxide (H2O2) was then added to the digested samples to oxidize Fe2+ ions to Fe3+ ions. The concentration of Fe2+ and Fe3+ ions in solution can be calculated by measuring the relaxation times before and after oxidation. The Fe3+/∑Fe ratio of slag samples was also investigated by wet chemistry and 57Fe Mössbauer spectroscopy and calculated by FactSage at the equilibrium state. The results obtained from different methods are consistent, indicating that this NMR relaxometry method is reliable.

Structure–property relations of sodium iron phosphate nuclear waste glasses: Effects of iron redox ratio and glass composition

AbstractIron phosphate glasses, known for their exceptional chemical durability and potential applicability in nuclear waste management, have gained significant attention over the years. The structures of these glasses are complicated by the coexistence of Fe3+ and Fe2+, which plays a crucial role in determining their structures and properties. This work uses molecular dynamics simulations to study the structural changes in Na2O–Fe2O3–P2O5 glasses with varying glass composition and Fe2+/Fe3+ redox ratio. It was found that the redox ratio and modifier contents significantly affected the short‐range and medium‐range orders in the glasses. Significant changes in the local environments around P5+ and Fe3+ were observed, as reflected by the bond distances and coordination numbers. Na+ cations are found to preferentially associate with Fe3+ (rather than Fe2+), whereas Fe2+ has stronger association with P5+ than Na+, confirming the structural role of Fe2+ as a glass modifier. The disruptions in P–O–P linkages upon increasing FeO suggest that FeO causes glass depolymerization. These glasses achieved higher connectivity with increasing ratios, conerting phosphorous Q2 to Q3 units and iron Q5 units to Q4 units. The decrease of nonbridging oxygen fractions with increasing ratios, through creating P–O–Fe linkages, is the main reason of enhanced network connectivity. Quantitative structure–property relationship analyses with different structural descriptors were used to correlate with measured properties. The analyses provided valuable insights into structure–property relationships, emphasizing the importance of choosing relevant energy parameters and defining glass network connectivity, particularly in Fnet descriptors. It was found the Fe–O–P linkage density exhibits strong correlations to measured dissolution rates, supporting the importance of these linkages in improving the chemical durability in iron phosphate glasses.

Safety and Efficacy of L-Glutamine in Reducing the Frequency of Acute Complications Among Patients with Sickle Cell Disease: A Randomized Controlled Study

Abstract Introduction Patients with sickle cell disease (SCD) suffer from recurrent and unpredictable episodes of acute pain due to vaso-occlusive crisis (VOC) which may require hospitalization and they also develop chronic persistent pain. Since pain usually begins in infancy and continues throughout life, preventing the genesis of pain is comparatively higher than treating the pain once it has been evoked VOC represents the cause of almost 95% of hospitalizations for patients with SCD. L-glutamine is an amino acid needed for the synthesis of the pyridines for nucleotides, including nicotinamide adenine dinucleotide (NAD) and glutathione, in addition to glutamate, and becomes essential during oxidative stress exposure. The NADH: [NAD+ + NADH] (redox) ratio in sickle red blood cells (RBCs) is lower than in normal RBCs which is related to oxidative stress, so L-glutamine availability is important in SCD. Although the FDA-approved it for preventing acute complications in SCD, still has many gaps in our understanding of its therapeutic implications. Aim To evaluate the safety and efficacy of L-glutamine in reducing the number of VOCs in patients with SCD. Methods This is an investigator-initiated single-center phase 4 interventional randomized controlled trial at Ain Shams Pediatric Hematology Unit. Sixty SCD participants, their mean age was 9.2 ± 3.7 years, they were 35 males and 25 females, were randomly assigned in a 1:1 ratio to receive glutamine at a dose of 0.3 gm/kg/dose twice daily orally (up to a maximum of 15 g/dose) for 24 weeks or the standard of care (SOC) without glutamine intake. Results Thirty-seven (62%) had HbSS with 38% had other sickle cell variants. All participants were receiving HU therapy and were on stable dose during the last three months prior to enrollment. Almost half of the patients (48%) were on chelation therapy with deferasirox. Thirty SCD patients were recruited in each group either received glutamine or SOC, both groups were comparable as regards demographic data. All patients in both arms had VOCs in the last year more than two. For the primary endpoint, there was a decreasing trend in the number, severity, and hospitalization of VOC in the glutamine arm and there was a significantly lower cumulative number of VOCs in the glutamine group (38.9%) than SOC (52.8%) (p = 0.008) and lower cumulative number of hospitalizations (p < 0.001). Conclusion Glutamine reduced the number of VOCs and hospitalizations for VOC in our cohort with SCD.

A Quantitative Comparison of 31P Magnetic Resonance Spectroscopy RF Coil Sensitivity and SNR between 7T and 10.5T Human MRI Scanners Using a Loop-Dipole 31P-1H Probe.

In vivo phosphorus-31 (31P) magnetic resonance spectroscopy (MRS) imaging (MRSI) is an important non-invasive imaging tool for studying cerebral energy metabolism, intracellular nicotinamide adenine dinucleotide (NAD) and redox ratio, and mitochondrial function. However, it is challenging to achieve high signal-to-noise ratio (SNR) 31P MRS/MRSI results owing to low phosphorus metabolites concentration and low phosphorous gyromagnetic ratio (γ). Many works have demonstrated that ultrahigh field (UHF) could significantly improve the 31P-MRS SNR. However, there is a lack of studies of the 31P MRSI SNR in the 10.5 Tesla (T) human scanner. In this study, we designed and constructed a novel 31P-1H dual-frequency loop-dipole probe that can operate at both 7T and 10.5T for a quantitative comparison of 31P MRSI SNR between the two magnetic fields, taking into account the RF coil B1 fields (RF coil receive and transmit fields) and relaxation times. We found that the SNR of the 31P MRS signal is 1.5 times higher at 10.5T as compared to 7T, and the power dependence of SNR on magnetic field strength (B0) is 1.9.

Shifting redox reaction equilibria on demand using an orthogonal redox cofactor.

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN+) as a noncanonical cofactor orthogonal to NAD(P)+. Here we present the development of Nox Ortho, a reduced NMN+ (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN+ ratio together with an NMN+-specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~103-106-fold cofactor specificity switch from NAD(P)+ to NMN+. We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).

Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems.

Neutrophils, the most abundant leukocytes in human peripheral circulation, are crucial for the innate immune response. They are typically quiescent but rapidly activate in response to infection and inflammation, performing diverse functions such as oxidative burst, phagocytosis, and NETosis, which require significant metabolic adaptation. Deeper insights into such metabolic changes will help identify regulation of neutrophil functions in health and diseases. Due to their short lifespan and associated technical challenges, the metabolic processes of neutrophils are not completely understood. This study uses optical metabolic imaging (OMI), which entails optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of single neutrophils. Primary human neutrophils were imaged in vitro under a variety of activation conditions and metabolic pathway inhibitors, while metabolic and functional changes were confirmed with mass spectrometry, oxidative burst, and NETosis measurements. Our findings show that neutrophils undergo rapid metabolic remodeling to a reduced redox state, followed by a shift to an oxidized redox state during activation. Additionally, single cell analysis reveals a heterogeneous metabolic response across neutrophils and donors to live pathogen infection ( Pseudomonas aeruginosa and Toxoplasma gondii ). Finally, consistent metabolic changes were validated with neutrophils in vivo using zebrafish larvae. This study demonstrates the potential of OMI as a versatile tool for studying neutrophil metabolism and underscores its use across different biological systems, offering insights into neutrophil metabolic activity and function at a single cell level.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

In vivo and in situ robust quantitative optical biopsy of hepatocellular carcinoma and metastasis based on two-photon autofluorescence imaging.

Two-photon autofluorescence (TPAF) imaging is able to offer precise cellular metabolic information with high spatiotemporal resolution, making it a promising biopsy tool. The technique is greatly hampered by the complexity of either the optical system or data processing. Here, the excitation wavelength was optimized to simultaneously excite both flavin adenine dinucleotide and nicotinamide adenine dinucleotide and eliminate the unexpected TPAF. The optical redox ratio (ORR) images were robustly achieved without additional calibration under the optimized single-wavelength excitation. The in vitro, ex vivo, and in vivo biopsy by the TPAF method were systematically studied and compared using hepato-cellular carcinoma and metastasis as examples. It was demonstrated that the proposed TPAF method simplified the optical system, improved the robustness of ORR, and enabled early-stage cancer diagnosis, showing distinguished advantages as compared with previous methods.

Metabolic changes in Toxoplasma gondii-infected host cells measured by autofluorescence imaging.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that infects warm-blooded vertebrates across the world. In humans, seropositivity rates of T. gondii range from 10% to 90% across communities. Despite its prevalence, few studies address how T. gondii infection changes the metabolism of host cells. In this study, we investigate how T. gondii manipulates the host cell metabolic environment by monitoring the metabolic response over time using noninvasive autofluorescence lifetime imaging of single cells, metabolite analysis, extracellular flux analysis, and reactive oxygen species (ROS) production. Autofluorescence lifetime imaging indicates that infected host cells become more oxidized and have an increased proportion of bound NAD(P)H compared to uninfected controls. Over time, infected cells also show decreases in levels of intracellular glucose and lactate, increases in oxygen consumption, and variability in ROS production. We further examined changes associated with the pre-invasion "kiss and spit" process using autofluorescence lifetime imaging, which also showed a more oxidized host cell with an increased proportion of bound NAD(P)H over 48 hours compared to uninfected controls, suggesting that metabolic changes in host cells are induced by T. gondii kiss and spit even without invasion.IMPORTANCEThis study sheds light on previously unexplored changes in host cell metabolism induced by T. gondii infection using noninvasive, label-free autofluorescence imaging. In this study, we use optical metabolic imaging (OMI) to measure the optical redox ratio (ORR) in conjunction with fluorescence lifetime imaging microscopy (FLIM) to noninvasively monitor single host cell response to T. gondii infection over 48 hours. Collectively, our results affirm the value of using autofluorescence lifetime imaging to noninvasively monitor metabolic changes in host cells over the time course of a microbial infection. Understanding this metabolic relationship between the host cell and the parasite could uncover new treatment and prevention options for T. gondii infections worldwide.

Metabolic profiling of single cells by exploiting NADH and FAD fluorescence via flow cytometry

ObjectiveThe metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. MethodsThe study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. ResultsOxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. ConclusionsTaken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

Characterization and classification of pathogenic bacteria using native fluorescence and spectral deconvolution.

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively.

Cellpose as a reliable method for single-cell segmentation of autofluorescence microscopy images.

Autofluorescence microscopy uses intrinsic sources of molecular contrast to provide cellular-level information without extrinsic labels. However, traditional cell segmentation tools are often optimized for high signal-to-noise ratio (SNR) images, such as fluorescently labeled cells, and unsurprisingly perform poorly on low SNR autofluorescence images. Therefore, new cell segmentation tools are needed for autofluorescence microscopy. Cellpose is a deep learning network that is generalizable across diverse cell microscopy images and automatically segments single cells to improve throughput and reduce inter-human biases. This study aims to validate Cellpose for autofluorescence imaging, specifically from multiphoton intensity images of NAD(P)H. Manually segmented nuclear masks of NAD(P)H images were used to train new Cellpose models. These models were applied to PANC-1 cells treated with metabolic inhibitors and patient-derived cancer organoids (across 9 patients) treated with chemotherapies. These datasets include co-registered fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD, so fluorescence decay parameters and the optical redox ratio (ORR) were compared between masks generated by the new Cellpose model and manual segmentation. The Dice score between repeated manually segmented masks was significantly lower than that of repeated Cellpose masks (p<0.0001) indicating greater reproducibility between Cellpose masks. There was also a high correlation (R2>0.9) between Cellpose and manually segmented masks for the ORR, mean NAD(P)H lifetime, and mean FAD lifetime across 2D and 3D cell culture treatment conditions. Masks generated from Cellpose and manual segmentation also maintain similar means, variances, and effect sizes between treatments for the ORR and FLIM parameters. Overall, Cellpose provides a fast, reliable, reproducible, and accurate method to segment single cells in autofluorescence microscopy images such that functional changes in cells are accurately captured in both 2D and 3D culture.

Investigating the relationship between hypoxia, hypoxia-inducible factor 1, and the optical redox ratio in response to radiation therapy

Investigating the relationship between hypoxia, hypoxia-inducible factor 1, and the optical redox ratio in response to radiation therapy

Ageing and Polypharmacy in Mesenchymal Stromal Cells: Metabolic Impact Assessed by Hyperspectral Imaging of Autofluorescence.

The impact of age on mesenchymal stromal cell (MSC) characteristics has been well researched. However, increased age is concomitant with increased prevalence of polypharmacy. This adjustable factor may have further implications for the functionality of MSCs and the effectiveness of autologous MSC procedures. We applied hyperspectral microscopy of cell autofluorescence-a non-invasive imaging technique used to characterise cytometabolic heterogeneity-to identify changes in the autofluorescence signals of MSCs from (1) young mice, (2) old mice, (3) young mice randomised to receive polypharmacy (9-10 weeks of oral therapeutic doses of simvastatin, metoprolol, oxycodone, oxybutynin and citalopram), and (4) old mice randomised to receive polypharmacy. Principal Component Analysis and Logistic Regression Analysis were used to assess alterations in spectral and associated metabolic characteristics. Modelling demonstrated that cells from young mice receiving polypharmacy had less NAD(P)H and increased porphyrin relative to cells from old control mice, allowing for effective separation of the two groups (AUC of ROC curve > 0.94). Similarly, cells from old polypharmacy mice were accurately separated from those from young controls due to lower levels of NAD(P)H (p < 0.001) and higher porphyrin (p < 0.001), allowing for an extremely accurate logistic regression (AUC of ROC curve = 0.99). This polypharmacy regimen may have a more profound impact on MSCs than ageing, and can simultaneously reduce optical redox ratio (ORR) and increase porphyrin levels. This has implications for the use of autologous MSCs for older patients with chronic disease.

Label-free single cell metabolic imaging of neutrophil activation

Abstract Upon activation, neutrophils combat infiltrating pathogens through a variety of effector functions, including oxidative burst, phagocytosis, and neutrophil extracellular traps (NETs). Metabolic pathways within neutrophils can be pharmacologically affected to modify immune outcomes. However, there is a lack of techniques to assess neutrophil metabolism in a label-free, non-destructive manner at single cell level. To address this, human neutrophil metabolism was investigated using two-photon microscopy of intrinsic fluorophores reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and flavin adenine dinucleotide, FAD. We image the optical redox ratio (ORR), [NAD(P)H / (NAD(P)H + FAD)] and the fluorescence lifetimes of NAD(P)H. Single-cell metabolic profiles of neutrophils were generated using an automatic cell segmentation pipeline. We found that activation of neutrophils either pharmacologically or by pathogen infection induced a significant metabolic change characterized by an increase in ORR and decrease in NAD(P)H lifetime, indicating an increase of the NAD(P)H pool, which suggests increases in glycolysis and the pentose phosphate pathway. Neutrophil activation was confirmed by measuring effector functions including oxidative burst. Importantly, identical metabolic changes were detected in the neutrophils of transgenic zebrafish larvae following activation, confirming the validity of these findings in vivo.