- 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
-
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
-
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
-
Digital scanned laser lighta??sheet fluorescence lifetime microscopy with widea??field timea??gated imaging
摘要： We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a sub-resolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimeters and can be utilized as a powerful and robust method for biomedical research, for example as a readout of protein-protein interactions via F?rster resonance energy transfer.
关键词： Fluorescence lifetime imaging,light-sheet fluorescence microscopy,time-resolved fluorescence imaging
更新于2025-09-23 15:19:57
-
Highly luminescent and stable CH3NH3PbBr3 quantum dots with 91.7% photoluminescence quantum yield: Role of guanidinium bromide dopants
摘要： Although perovskite quantum dots (PQDs) have received considerable attention, defects in PQDs can significantly degrade the properties and device performance. In this study, we report on an effective strategy for synthesizing highly luminescent CH3NH3PbBr3 quantum dots (QDs) by a simple doping. To remove such defects, guanidinium bromide (GuBr) was doped into the CH3NH3PbBr3 QDs synthesized by the ligand-assisted reprecipitation technique. From XRD and TEM studies, the doping of GuBr into the QD lattices was verified. In addition, the surfaces of PQDs with and without GuBr dopants were analyzed by XPS to trace the metallic Pb acting as a major recombination center. The GuBr doping resulted in the size uniformity of QDs and effectively eliminated defects and metallic Pb, which enhanced the photoluminescence quantum yield (PLQY) through the inhibition of the non-radiative recombination pathway. Furthermore, the recombination dynamics in the QDs were examined by using time-resolved photoluminescence and fluorescence lifetime imaging to verify the role of GuBr dopants. By optimizing the amount of GuBr doping, the CH3NH3PbBr3 QDs with strong green emission achieved a maximum PLQY of 91.7%.
关键词： Photoluminescence quantum yield,Fluorescence lifetime imaging,CH3NH3PbBr3 quantum dots,Guanidinium bromide,Recombination centers
更新于2025-09-23 15:19:57
-
Distinct metabolic profiles in Drosophila sperm and somatic tissues revealed by two-photon NAD(P)H and FAD autofluorescence lifetime imaging
摘要： Metabolic profiles vary across all levels of biological diversity, from cells to taxa. Two-photon fluorescence lifetime imaging microscopy (FLIM) facilitates metabolic characterisation of biological specimens by assaying the intrinsic autofluorescence of the ubiquitous coenzymes NAD(P)H and FAD. the potential of this method for characterising the diversity of organismal metabolism remains largely untapped. Using FLIM in Drosophila melanogaster, we show tissue-specificity in fluorescence lifetime that reflects variation in redox patterns. In particular, sperm cells exhibited elevated glycolysis relative to other tissues. We also show that sperm metabolism is phenotypically plastic: compared to male-stored sperm, sperm stored in the female’s storage organ showed a substantial reduction in the protein-bound FAD lifetime fraction but no change in the NAD(P)H profile. This study represents the first ex vivo investigation of sperm metabolism using FLIM.
关键词： two-photon fluorescence lifetime imaging microscopy,FAD,Drosophila melanogaster,NAD(P)H,glycolysis,sperm metabolism,OxPHOS
更新于2025-09-12 10:27:22
-
A Quantum Dot-Based FLIM Glucose Nanosensor
摘要： In the last few years, quantum dot (QD) nanoparticles have been employed for bioimaging and sensing due to their excellent optical features. Most studies have used photoluminescence (PL) intensity-based techniques, which have some drawbacks, especially when working with nanoparticles in intracellular media, such as fluctuations in the excitation power, fluorophore concentration dependence, or interference from cell autofluorescence. Some of those limitations can be overcome with the use of time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM) techniques. In this work, CdSe/ZnS QDs with long decay times were modified with aminophenylboronic acid (APBA) to achieve QD-APBA conjugates, which can act as glucose nanosensors. The attachment of the boronic acid moiety on the surface of the nanoparticle quenched the PL average lifetime of the QDs. When glucose bonded to the boronic acid, the PL was recovered and its lifetime was enhanced. The nanosensors were satisfactorily applied to the detection of glucose into MDA-MB-231 cells with FLIM. The long PL lifetimes of the QD nanoparticles made them easily discernible from cell autofluorescence, thereby improving selectivity in their sensing applications. Since the intracellular levels of glucose are related to the metabolic status of cancer cells, the proposed nanosensors could potentially be used in cancer diagnosis.
关键词： fluorescence lifetime imaging,intracellular sensing,quantum dots,nanoparticles,glucose,photoluminescence
更新于2025-09-11 14:15:04
-
Fluorescence Lifetime-Based Tumor Contrast Enhancement Using an EGFR Antibody–Labeled Near-Infrared Fluorophore
摘要： Purpose: Imaging techniques for highly specific detection of cancer cells in vivo can have applications ranging from preclinical drug discovery studies to clinical cancer diagnosis and surgical therapy. Although fluorescence imaging using cancer-targeted antibodies has shown promise, nonspecific probe accumulation in tissue results in significant background fluorescence, reducing detection sensitivity using traditional intensity–based continuous-wave (CW) fluorescence imaging. Here we demonstrate that fluorescence lifetime (FLT) imaging can provide significant tumor contrast enhancement over CW intensity in preclinical models of human breast cancer. Experimental Design: Mice bearing MDA-MB-231 tumors were injected with anti-EGFR antibody conjugated to the fluorescent dye IRDye 800CW (anti-EGFR-800). Time domain fluorescence imaging was performed in vivo and in situ up to 48 hours after dye injection. Results: Mice injected with anti-EGFR-800 showed a significantly longer FLT (0.7 ± 0.03 ns) compared with the FLT of nonspecific probe uptake in liver (0.63 ± 0.05 ns), providing a dramatic improvement in sensitivity and specificity compared with CW intensity. IgG antibody–conjugated IRDye 800CW did not show an increased FLT compared with normal tissue, suggesting that the FLT increase of anti-EGFR-800 in tumors was associated with receptor expression. Using serial surgery, we show that FLT allows the detection of smaller residual tumors in the surgical bed than possible using CW intensity. Conclusions: Our data suggest that FLT can significantly enhance tumor contrast using fluorescently labeled antibodies, thereby accelerating the efficient clinical application of these probes for margin assessment in image-guided surgery and for highly specific detection of tumor receptors in vivo.
关键词： near-infrared fluorophore,EGFR,fluorescence lifetime imaging,image-guided surgery,tumor contrast
更新于2025-09-11 14:15:04
-
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