- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
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
-
Measuring the interaction of transcription factor Nrf2 with its negative regulator Keap1 in single live cells by an improved FRET/FLIM analysis
摘要： Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. Detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualise and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured Instrument Response Function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
关键词： FRET,live cell imaging,fluorescence lifetime,FLIM,sfGFP,protein-protein interaction,global binning,Keap1,Instrument Response Function,Nrf2
更新于2025-11-21 11:08:12
-
A Study on the Solvent Vapor Induced Film Formation of PS/AgNPs Composites
摘要： In this study, pyrene (P) tagged polystyrene (PS) latex dispersions and silver nanoparticles (AgNPs) were mixed at various weight fractions in the range between 0 and 50 wt%. The prepared mixtures were dropped on the glass substrates by considering drop casting method and were dried at the room temperature. The resultant powder films were then exposed to solvent vapor to monitor how film formation and morphological behaviors of PS/AgNPs composites are altered. Film formation behavior of composites was assessed via fast transient fluorescence (FTRF) which measures the ?uorescence lifetimes of P from its decay traces during vapor exposure process. It was observed that pyrene lifetimes decreased as vapor exposure time, t increased. A Stern–Volmer kinetic analysis was used for low quenching ef?ciencies to interpret the decrease in pyrene lifetimes. UV-Vis (UVV) technique was employed to monitor optical transparency of the films. In the range of 0-20 wt% of AgNPs content, smooth and transparent films were obtained. However, above this range, the films were seen that they have low transparency and poor film formation since the increment in AgNPs content was lead to aggregations. The Prager–Tirrel model was employed to the FTRF data to obtain back-and-forth frequencies, ν, of the reptating PS chains during vapor induced ?lm formation process. SEM images of the samples were taken after film formation process is completed and were found to be consistent with optical and fluorescence quenching data.
关键词： Polystyrene latex,film formation,reptation frequency,fluorescence lifetime,nanocomposites,crossing-density,Silver nanoparticles,vapor-induced
更新于2025-09-23 15:23:52
-
Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence
摘要： Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this study, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over fluorophores, reduced fluorescent time. Two endogenous nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.
关键词： fluorescence lifetime,fixed cells,Autofluorescence,mounting medium
更新于2025-09-23 15:23:52
-
A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM
摘要： Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.
关键词： super-resolution,SPAD array,fluorescence lifetime imaging,confocal microscopy,image scanning microscopy
更新于2025-09-23 15:23:52
-
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
-
Fluorescent Nanodiamonds || Producing Fluorescent Nanodiamonds
摘要： Natural diamonds in colors are commonly known as fancies, or fancy color diamonds, in gemstone industries. They are rare, beautiful, and some even carry impressive price tags in the jewelry market. By comparison, micro‐ and nanoscale diamond powders are low in price, with or without colors and fluorescent or not. These powders have been used as abrasives for grinding and polishing purposes since ancient time, mainly because of their extraordinary hardness. Little or no attention has been paid over the centuries to other properties of nanodiamonds such as their innate biocompatibility and light‐emitting capability. The invention of fluorescent nanodiamond (FND) in 2005 has revolutionized the field, opening a new area of research and development with diamonds. Experiments with FNDs in the last decade have demonstrated various promising applications of surface‐functionalized FNDs in diversified fields, ranging from physics and chemistry to biology and medicine. It is worthy of noting that as originated from the discovery of Radium by Marie Sk?odowska Curie (Section 3.2), FNDs may very well be called Madame Curie’s gemstones, valued appropriately as a scientist’s best friend.
关键词： fluorescent nanodiamonds,magnetically modulated fluorescence,fluorescence lifetime,size reduction,FND,ion irradiation,H3 centers,nitrogen-vacancy centers,electron irradiation,NV centers
更新于2025-09-23 15:21:01
-
Increasing fluorescence lifetime for resolution improvement in STED nanoscopy
摘要： Super-resolution microscopy (SRM) has had a substantial impact on the biological sciences due to its ability to observe tiny objects less than 200 nm in size. Stimulated emission depletion (STED) microscopy represents a major category of these SRM techniques that can achieve diffraction-unlimited resolution based on a purely optical modulation of fluorescence behaviors. Here, we investigated how the laser beams affect fluorescence lifetime in both confocal and STED imaging modes. The results showed that with increasing illumination time, the fluorescence lifetime in two kinds of fluorescent microspheres had an obvious change in STED imaging mode, compared that in confocal imaging mode. As a result, the reduction of saturation intensity induced by the increase of fluorescence lifetime can improve the STED imaging resolution at the same depletion power. The phenomenon was also observed in Star635P labeled human Nup153 in fixed HeLa cells, which can be treated as a reference for the synthesis of fluorescent labels with the sensitivity to the surrounding environment for resolution improvement in STED nanoscopy.
关键词： confocal microscopy,super-resolution,fluorescence lifetime,fluorescence microscopy
更新于2025-09-23 15:21:01
-
Concentration quenching resistant donor-acceptor molecular structure for high efficiency and long lifetime thermally activated delayed fluorescent organic light-emitting diodes via suppressed non-radiative channel
摘要： A molecular design having t-butyl groups surrounding a donor-acceptor type core structure was developed as an approach to obtain high external quantum efficiency by suppressing concentration quenching effect caused by strong intermolecular interaction. The donor-acceptor type core structure was surrounded by six t-butyl groups to separate the donor-acceptor core structure between molecules. A heptazine acceptor and a diphenylamine donor based thermally activated delayed fluorescent emitter protected by the multiple t-butyl units achieved maximum external quantum efficiency of 32.6% at 1% doping concentration and 23.0% even at a high doping concentration of 20% by reducing concentration quenching effect. Furthermore, the lifetime of the thermally activated delayed fluorescent devices was also improved relative to that of the previous emitter with the same acceptor. The external quantum efficiency and device lifetime are better than any other results reported in the orange TADF OLEDs.
关键词： Thermally activated delayed fluorescence,Lifetime,T-butyl,Organic light-emitting diodes,High efficiency
更新于2025-09-23 15:21:01
-
Nitrogen-doped graphene quantum dots prepared by electrolysis of nitrogen-doped nanomesh graphene for the fluorometric determination of ferric ions
摘要： Nitrogen-doped graphene quantum dots (N-GQDs) were synthesized by direct electrolysis of a carbon cloth electrode coated with nitrogen-doped nanomesh graphene (NG) in high yield (~ 25%). The N-GQDs emit intense blue fluorescence with a quantum yield (QY) of 10% ± 3%. Meanwhile, the N-GQDs are rich in hydroxyl, carboxyl, basic pyridinic nitrogen, and nitro groups, which are conducive to strengthen the interaction between N-GQDs and Fe3+ for highly sensitive determination of Fe3+ ions. Specifically, the determination for Fe3+ was conducted at different concentrations of N-GQD solution with a wide linear range of 10–1000 μM (150 μg·mL?1) and a low detection limit of 0.19 μM (10 μg·mL?1). Moreover, the fluorescence quenching mechanism illustrated that the functional groups generated by electrochemical oxidation enhanced the interaction of N-GQDs and Fe3+, and the narrow band gap (2.83 eV) of N-GQDs accomplished electron transfer from N-GQDs to Fe3+ easily.
关键词： Fluorescence lifetime,Band gap,Dynamic quenching,Carbon cloth electrode,Electrochemical oxidation
更新于2025-09-23 15:21:01