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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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A high-affinity fluorescence probe for copper(II) ions and its application in fluorescence lifetime correlation spectroscopy
摘要： Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper’s redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = ? 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms.
关键词： BODIPY,Single molecule detection,FLIM,Alzheimer,ATCUN motif,Parkinson,DAHK
更新于2025-11-21 11:24:58
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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
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Design, synthesis and photophysical studies of improved xanthene dye to detect acetate
摘要： Development of biomarkers of analytes with interest in clinic is an important field of study. In this work, we synthesized and analyzed the new fluorescent acetate-biomarker, Iso-PG. The mechanism of detection is the acetate buffer mediated proton transfer reaction. The rate constants involved were obtained, and we measured the change in the fluorescence lifetime produced as a consequence of the presence of acetate in the medium. Finally, we checked its potential use as acetate biomarker in synthetic serum.
关键词： FLIM,Acetate detection,Biomarkers,New dye
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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Layer-dependent signatures for exciton dynamics in monolayer and multilayer WSe2 revealed by fluorescence lifetime imaging measurement
摘要： Two-dimensional (2D) transition-metal dichalcogenide (TMD) materials have aroused noticeable interest due to their distinguished electronic and optical properties. However, little is known about their complex exciton properties together with the exciton dynamics process which have been expected to influence the performance of optoelectronic devices. The process of fluorescence can well reveal the process of exciton transition after excitation. In this work, the room-temperature layer-dependent exciton dynamics properties in layered WSe2 are investigated by the fluorescence lifetime imaging microscopy (FLIM) for the first time. This paper focuses on two mainly kinds of excitons including the direct transition neutral excitons and trions. Compared with the lifetime of neutral excitons (< 0.3 ns within four-layer), trions possess a longer lifetime (~ 6.6 ns within four-layer) which increases with the number of layers. We attribute the longer-lived lifetime to the increasing number of trions as well as the varieties of trion configurations in thicker WSe2. Besides, the whole average lifetime increases over 10% when WSe2 flakes added up from monolayer to four-layer. This paper provides a novel tuneable layer-dependent method to control the exciton dynamics process and finds a relatively longer transition lifetime of trions at room temperature, enabling to investigate in the charge transport in TMD-based optoelectronics devices in the future.
关键词： two-dimensional (2D) WSe2,fluorescence lifetime,fluorescence lifetime imaging microscopy (FLIM),exciton dynamics,density functional theory (DFT)
更新于2025-09-23 15:19:57
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Green emitted CdSe@ZnS quantum dots for FLIM and STED imaging applications
摘要： Inorganic quantum dots (QDs) have excellent optical properties, such as high fluorescence intensity, excellent photostability and tunable emission wavelength, etc., facilitating them to be used as labels and probes for bioimaging. In this study, CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope (FLIM) and stimulated emission depletion (STED) nanoscopy imaging. The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW. The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser (27.5 mW) irradiation. Furthermore, the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes. Meanwhile, Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM, and their fluorescence lifetime was around 3.1 ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.
关键词： CdSe@ZnS QDs,living cells,STED,FLIM
更新于2025-09-19 17:13:59
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The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy
摘要： Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy F?rster resonance energy transfer (fLiM-fRet) and stimulated emission depletion (SteD) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.
关键词： FLIM-FRET,living cells,magnetotactic bacteria,STED microscopy,protein mechanics
更新于2025-09-12 10:27:22
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Metabolic imaging with the use of?fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in?mouse oocytes
摘要： To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction. Experimental study. Academic research laboratories. None. Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n ? 52) were compared with wild-type (WT) oocytes (n ? 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n ? 29) were compared with oocytes from young mice (12 weeks old; n ? 35) as a model of mild oocyte dysfunction. FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (t1) and long (t2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety. In Clpp-knockout oocytes compared with WT, FAD t1 and t2 were longer and I was higher, NADH t2 was longer, and F was lower. In old oocytes compared with young ones, FAD t1 was longer and I was lower, NADH t1 and t2 were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates. FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.
关键词： Mitochondria,aging,mitochondrial unfolded protein response,fluorescence lifetime imaging microscopy,CLPP,FLIM,oocyte
更新于2025-09-10 09:29:36
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Changing Times: Fluorescence-Lifetime Analysis of Amyloidogenic SF-IAPP Fusion Protein
摘要： In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils’ detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.
关键词： FLIM,IAPP,amyloid-like fibrils,green fluorescent protein,atomic force microscopy,conformational diseases
更新于2025-09-09 09:28:46