Search Results (281)

Neurodegenerative diseases feature diverse pathological protein aggregates, including Lewy bodies in Alzheimer’s disease (AD) and skein-like filaments in amyotrophic lateral sclerosis (ALS). The physical mechanisms underlying this morphological diversity remain unclear. Here, we demonstrate that aggregation of the prion-like domain of hnRNPA1 (A1PrD), implicated in AD and ALS, is driven by solution composition and phase transition dynamics. Utilizing 3D timelapse and fluorescence lifetime imaging microscopy, we show that solution conditions modulate phase separation, gelation, and fibrillation, resulting in distinct structures such as fibril, gel, and starburst morphologies. Homotypic and heterotypic interactions between A1PrD and RNA were observed to shift the balance between pathological and physiological condensates. Importantly, amyloid-rich starbursts displayed prion-like infection capabilities toward amyloid-poor condensates. Our findings highlight how the interplay between solution composition and kinetic balances of liquid-liquid phase separation, gelation, and fibrillation shapes the diverse pathological aggregate morphologies characteristic of neurodegenerative diseases. Full article

Background/Objectives: Early embryonic arrest during the cleavage stage (days 2–4) accounts for a substantial proportion of developmental failure in in vitro fertilization. This phenomenon remains poorly understood at the molecular level, even in chromosomally normal embryos identified by preimplantation genetic testing. This review aims to redefine cleavage-stage arrest from a passive energy deficit to a checkpoint-regulated endpoint caused by inadequate coordination among metabolism, transcriptome integrity, and stress-response pathways. Methods: We integrate evidence from long-read transcriptomics, metabolomics, epigenetics, and immunobiology relevant to pre-blastocyst development. These data are assembled into a unifying mechanistic framework and a clinically oriented stratification model, together with candidate multimodal readouts for early classification. Results: We propose a three-axis model linking: (i) metabolic–epigenetic insufficiency, including defective histone lactylation and impaired alpha-ketoglutarate-dependent DNA demethylation; (ii) isoform-level abnormalities, including intron retention and retrotransposon activation within a hidden transcriptomic landscape better resolved by long-read sequencing; and (iii) stress-related immune signaling, in which NLRP7 links alternative splicing and DNA-damage-response dysfunction with mitochondrial stress and p53-associated arrest. Within this framework, we distinguish three molecular arrest states: an early transition failure marked by defective maternal-to-embryonic reprogramming and severe splicing disruption; a metabolically quiescent state that may retain a limited rescue window; and a later stress-associated state characterized by senescence-like features, oxidative stress, and broad transcriptomic and genomic instability. Conclusions: Early embryo arrest should no longer be viewed as a nonspecific developmental failure, but as a mechanistically stratifiable condition with distinct metabolic, transcriptomic, and stress-associated trajectories. A diagnostic platform combining fluorescence lifetime imaging microscopy, long-read sequencing, and digital polymerase chain reaction may improve early mechanistic classification, help identify embryos with possible reversibility, and reduce uncertainty in embryo selection during in vitro fertilization. Full article

Molecular interactions underpin the functioning of the living cell. Molecules exist in distinct quaternary structural forms, associate with molecular partners in signaling cascades, form transient quinary interactions, localize in membrane domains, and cluster in membrane-less condensates. Measuring the concentration, size, and dynamics of these molecular assemblies remains an enduring biophysical challenge, particularly in cells, where heterogeneity is the rule rather than the exception. Orthogonal signals derived from fluorescence lifetime, fluorescence fluctuations, and fluorescence polarization provide valuable metrics for probing interactions and environments, concentration and size, and rotational dynamics, respectively. This paper combines fluorescence lifetime imaging microscopy with image correlation analysis and polarization to determine the concentrations, brightness, lifetime, and rotational correlation time of different fluorescent states. A two-population model is examined as a prototypical example of a heterogeneous system. The analysis is illustrated on a simple fluorescence model system, where cluster densities, relative brightnesses, lifetimes, and rotational correlation times are extracted. Full article

The effect of the confinement of fluorophores (rhodamine 6G) in nano-cavities of porous 3D sculptured coatings made by glancing-angle deposition (GLAD) was investigated by fluorescence-lifetime imaging microscopy (FLIM). Shortening of fluorescence/ photoluminescence lifetime by ∼10% was observed from the dye-permeated (in liquid) structure; however, there was no rotational hindrance of dye molecules. When dried, a strong rotational hindrance $89\%$ was observed for the orientation along the ordinary optical axis (slow-axis), and the hindrance was smaller than $57\%$ for the extraordinary direction (fast axis). Light-intensity distribution inside the nano-structure with a form birefringence was numerically modeled using plane-wave illumination and a dipole source. Nanoscale localization of light intensity due to dipole nature $I\sim 1/radiu{s}^6$ and boundary conditions for E-field allows efficient energy deposition inside the region of lower refractive index (nanogaps). Full article

Lignin from Phaleria macrocarpa (Mahkota Dewa) fruit, a bioactive-rich cultivated medicinal biomass, was employed as a renewable precursor for lignin quantum dots (LQDs). A simple, aqueous, catalyst-free two-step route (lignin to lignin nanoparticles to LQDs) is demonstrated, enabling the valorization of non-food lignin into photoluminescent nanomaterials. The resulting LQDs were predominantly amorphous with short-range graphitic ordering and a narrow particle size distribution (3–5 nm). Structural and chemical analyses indicated a partially graphitized carbon framework enriched with oxygenated surface functionalities, which is consistent with their bright blue–green emission (λ_em of 490 nm; average fluorescence lifetime of 4.51 ns). Hydrothermal carbonization induced a blue shift in the UV–Vis absorption profile, resulting in a main band at 288 nm with a shoulder at 312 nm. The LQDs exhibited high cytocompatibility toward L929 mouse fibroblasts (93.1 ± 6.5% viability at 24 h) and were readily internalized by cells, facilitating green fluorescence live-cell imaging as a proof-of-concept. Antibacterial activity was observed against both Gram-positive and Gram-negative strains, supporting dual biofunctional performance. Overall, this study established a green and scalable route for converting P. macrocarpa fruit lignin into multifunctional LQDs, with potential applications in circular-bioeconomy such as antimicrobial/active coatings and optical sensing in agro-industrial contexts. Full article

Culturing cells and micro-tissue samples in 3D bio-scaffolding structures is gaining popularity; however, precise control of tissue micro-environment in such systems remains challenging. We describe a family of new hybrid bio-scaffolds with 3D O₂-sensing ability, produced by simple means from readily available bio-scaffolding and O₂-sensing materials. Three different types of phosphorescent O₂-sensing materials—polymeric microparticles (MPs), supramolecular probe MitoXpress and nanoparticulate probes NanO2 and Nano-IR (NPs)—were integrated in Matrigel and agarose scaffolding materials and evaluated. Key working characteristics of such hybrid scaffolds, including heterogeneity, stability, cytotoxicity, optical signals and O₂-sensing properties, ease of fabrication and use, were compared. The results show superiority of the Matrigel hybrids with NanO2 and Nano-IR probes. Demonstration experiments were conducted with HCT116 cells and individual spheroids derived from these cells, culturing them in the Matrigel–NP hybrid scaffolds and monitoring oxygenation and local O₂ gradients on a time-resolved fluorescence plate reader and by phosphorescence lifetime imaging microscopy (PLIM). Full article

Multicomponent mixed lanthanide oxide (MMLO) nanoparticles possess considerable potential as multimodal imaging agents because they integrate diverse excellent optical and magnetic properties within a single nanoparticle. Herein, we present triply and quadruply mixed lanthanide oxide nanoparticles, namely, gadolinium (Gd)/dysprosium (Dy)/europium (Eu) oxide (GDEO), Gd/Dy/terbium (Tb) oxide (GDTO), and Gd/Dy/Eu/Tb oxide (GDETO) nanoparticles. Gd³⁺ can strongly induce positive (T₁) contrast in magnetic resonance imaging (MRI), Dy³⁺ and Tb³⁺ can generate negative (T₂) contrast in MRI, and Eu³⁺ and Tb³⁺ emit visible photons that are applicable to fluorescence imaging (FI). All the nanoparticles were grafted with hydrophilic, biocompatible polyacrylic acid (PAA) to enhance colloidal stability and biocompatibility and further grafted with small amounts of an organic photosensitizer, 2,6-pyridinedicarboxylic acid (PDA), to obtain a high absolute fluorescent quantum yield (QY) with an extended fluorescent lifetime (τ). All PAA-MMLO and PAA/PDA-MMLO nanoparticles exhibited nearly monodispersed particle-size distributions with average particle diameters of ~2 nm and displayed considerably higher longitudinal (r₁) and transverse (r₂) water proton spin relaxivities than commercial molecular MRI contrast agents. The PAA/PDA-GDEO, PAA/PDA-GDTO, and PAA/PDA-GDETO nanoparticles exhibited high absolute QYs of 45, 29, and 61%, respectively, and long τ values of 1–2 ms, making them suitable for time-delayed noise-free fluorescence signal detection. These findings confirm the high potential of PAA-MMLO nanoparticles as T₁ and/or T₂ MRI contrast agents and PAA/PDA-MMLO nanoparticles as both T₁ and/or T₂ MRI and FI agents. Full article

The palette of the fluorogen-activating protein FAST expanded into the far-red region by the development of a novel fluorogen, HBTR-3,5-DOM. This was achieved through a C=O to C=S substitution in the classic hydroxybenzylidene-rhodanine core, which induced a bathochromic shift of over 100 nm. The complexes of HBTR-3,5-DOM with FAST variants pFAST and F62L are characterized by absorption and emission maxima at 640–650 nm and ~670 nm, respectively, and are found to exhibit distinct fluorescence lifetimes. The fluorogen is successfully applied in genetically encoded live-cell imaging together with these FAST variants for various subcellular structures. Furthermore, its potential for multiplexed imaging is demonstrated by the simultaneous discrimination of two targeted proteins using fluorescence lifetime imaging microscopy (FLIM). Full article

We report on the realization of a platform for trapping and manipulating individual ⁸⁸Sr atoms in optical tweezers. A first cooling stage based on a blue shielded magneto-optical trap (MOT) operating on the ${|}^1{\mathrm{S}}_0⟩\to {|}^1{\mathrm{P}}_1⟩$ transition at $461 \mathrm{n}\mathrm{m}$ enables us to trap approximately 4 × 10⁶ atoms at a temperature of 6.8 mK. Further cooling is achieved in a narrow-line red MOT using the ${|}^1{\mathrm{S}}_0⟩\to {|}^3{\mathrm{P}}_1⟩$ intercombination transition at 689 nm, bringing 5 × 10⁵ atoms down to $5\mu \mathrm{K}$ and reaching a density of 4 × 10¹⁰ cm³. Atoms are then loaded into 813 nm tweezer arrays generated by crossed acousto-optic deflectors and tightly focused onto the atoms with a high-numerical-aperture objective. Through light-assisted collision processes we achieve the collisional blockade, which leads to single-atom occupancy with a probability of about 50%. The trapped atoms are detected via fluorescence imaging with a fidelity of $99.986\left(6\right)\%$, while maintaining a survival probability of $97\left(2\right)\%$. The release-and-recapture measurement provides a temperature of $12.92\left(5\right)\mu \mathrm{K}$ for the atoms in the tweezers, and the ultra-high-vacuum environment ensures a vacuum lifetime higher than 7 min. These results demonstrate a robust alkaline-earth tweezer platform that combines efficient loading, cooling, and high-fidelity detection, providing the essential building blocks for scalable quantum simulation and quantum information processing with Sr atoms. Full article

Background/Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor with dismal prognosis and limited treatment options. Immunotherapy, including personalized approaches using tumor-infiltrating lymphocytes (TILs) and allogeneic natural (NK) or engineered killer cells (chimeric antigen receptor NK, NK-CAR), and oncolytic viruses (OV), has shown some potential in GBM. Combining different therapeutic strategies may enhance treatment efficacy. Here, we present a xenograft GBM mouse model with multiparametric detection for various immunotherapy research applications. Methods: In a xenograft GBM NOD-Prkdcs scid Il2rgem1/Smoc (NSG) mouse model based on orthotopic transplantation of patient-derived GBM cultures retaining tumor heterogeneity, intravenous and intratumor immunotherapeutic interventions by TIL and OV therapy were performed. Xenograft engraftment was evaluated using intravital MRI; delivery of OV and TILs to the tumor and changes in the tumor and peritumoral space were assessed using intravital confocal microscopy; and metabolic and structural changes in the tumor and peritumoral environment were assessed via fluorescence lifetime imaging microscopy (FLIM) and optical coherence tomography (OCT). The intravital imaging data were compared with the results of preliminary and final histological and immunocytochemical data. Results: Both OV and TILs demonstrated tumor-specific targeting and delivery across the blood–brain barrier. Further, we showed that in this model the xenograft response to both therapeutic treatments can be assessed using FLIM and OCT. Conclusions: Overall, this work presents an optimized mouse model suitable for assessing the effect of combined TIL immunotherapy and OV on GBM in translational studies. Full article

Back focal plane (BFP) imaging has emerged as a widely used technique for investigating various nanoscale optical devices. The ability to provide the full angular distribution of emitted light has enabled the engineering of precise radiation patterns, enabling new advances in nanophotonics. Continuous improvements in the BFP imaging technique, including wavelength, polarization, and phase-resolved signal detection, have allowed us to gain crucial insights into the various optical and material properties of nanophotonic devices. In this study, we introduce a fluorescence lifetime-resolved BFP imaging configuration, which uses a spatial filtering technique in the Fourier plane to discriminate between different emission directions. Uniform silver film (45 nm) with a PMMA matrix layer of about 20 nm containing Rhodamine 6G fluorescent molecular dye was prepared and measured using total internal reflection ellipsometry (TIRE). A coupled oscillator model was used, and strong coupling was observed with a coupling strength of 160 meV. Time-correlated single-photon counting was used for the estimation of fluorescence lifetime in the sub-nanosecond regime, and a direction-dependent lifetime was observed in the BFP imaging configuration. This modified fluorescence-lifetime-resolved BFP microscopy method is essential for directly correlating the collective quantum dynamics (lifetime/decay rate) with the far-field radiation pattern (angle/coherence). It offers a critical tool for designing and optimizing quantum nanophotonic devices, such as polariton-based components and highly directional single-photon emitters, where controlling both excited-state dynamics and spatial coherence is paramount. Full article

Solar lentigo is a significant dermatological concern affecting individuals of different genders and ethnicities. Its pathogenesis is primarily attributed to chronic ultraviolet (UV) exposure, increased melanogenesis, and disrupted epidermal turnover, leading to the development of hyperpigmented lesions. A major challenge in solar lentigo research is acquiring viable skin tissue, which is crucial for understanding the dynamics of the cellular microenvironment. In the present study, we sought to establish a non-invasive in vivo measurement technique to visualize cellular dynamics associated with solar lentigo. Utilizing fluorescence lifetime imaging microscopy (FLIM), we quantified the decay of NAD(P)H fluorescence lifetime and observed a reduction in oxidative phosphorylation (OXPHOS) activity in solar lentigo lesions compared to adjacent non-lesional skin. To determine whether the observed reduction in OXPHOS activity was due to excessive melanin accumulation in keratinocytes, we developed a melanin deposition model and examined the pleiotropic alterations occurring in keratinocytes following the phagocytosis of excessive melanin. Our findings indicate that excessive melanin deposition downregulates OXPHOS in differentiating keratinocytes and induces senescence-associated phenotypes characterized by perturbed cell cycle progression, increased cell size and aneuploidy, and the secretion of inflammatory mediators in proliferating keratinocytes. Collectively, our results implicate a solar lentigo-specific senescence mechanism driven by excessive melanin accumulation in keratinocytes, providing new insights about the intrinsic modulators of the pathological condition. Full article

Alpha-synuclein (αsyn) misfolding and aggregation underlie several neurodegenerative disorders, including Parkinson’s disease. Early oligomeric intermediates are particularly toxic yet remain challenging to detect and characterize within cellular systems. Here, we employed the luminescent conjugated oligothiophene h-FTAA to investigate early aggregation events of human wildtype (huWT) and A53T-mutated αsyn (huA53T) both in vitro and in HEK293 cells stably expressing native human-αsyn. Comparative fibrillation assays revealed that h-FTAA detected αsyn aggregation with higher sensitivity and earlier onset than Thioflavin T, with the A53T variant displaying accelerated fibrillation. HEK293 cells stably expressing huWT- or huA53T-αsyn were exposed to respective pre-formed fibrils (PFFs), assessed via immunocytochemistry, h-FTAA staining, spectral emission profiling, and fluorescence lifetime imaging microscopy (FLIM). Notably, huA53T PFFs promoted earlier aggregation patterns and yielded narrower fluorescence lifetime distributions compared with huWT PFFs. Spectral imaging showed h-FTAA emission maxima (~550–580 nm) red-shifted and broadened in cells along with variable lifetimes (0.68–0.87 ns), indicating heterogeneous aggregate conformations influenced by cellular milieu. These findings demonstrate that h-FTAA is useful for distinguishing early αsyn conformers in living systems and, together with stable αsyn-expressing HEK293 cells, offers a platform for probing early αsyn morphotypes. Taken together, this opens for further discovery of biomarkers and drugs that can interfere with αsyn aggregation. Full article

Protein–protein interactions (PPIs) play a crucial role in various biological processes, including signal transduction, transcriptional regulation, and metabolic pathways. Over the years, many methods have been developed to study PPIs, such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), pull-down assays, and surface plasmon resonance (SPR). However, each of these techniques has its own limitations, including false positives, a lack of specific binding partners, and restricted interaction zones. Fluorescence resonance energy transfer (FRET) has emerged as a powerful technique for investigating PPIs, offering several advantages over traditional methods. Recent advancements in fluorescence microscopy have further enhanced its application in PPI studies. In this review, we summarize recent developments in FRET-based approaches and their applications in PPIs research over the past five years, including conventional FRET, time-resolved FRET (TR-FRET), fluorescence lifetime imaging microscopy-FRET (FLIM-FRET), single-molecule FRET (smFRET), fluorescence cross-correlation spectroscopy FRET (FCCS-FRET), and provide guidance on selecting the most appropriate method for PPIs studies. Full article

Zn-doped carbon dots (Zn@C-210 calcination temperature at 210 °C and Zn@C-260 calcination temperature at 260 °C) were synthesized via an in situ calcination method using zinc citrate complexes as precursors, aiming to investigate the mechanisms of their distinctive fluorescence properties. A range of analytical methods were employed to characterize these nanomaterials. The mechanism study revealed that the coordination structure of Zn-O, formed through zinc doping, can induce a metal–ligand charge-transfer effect, which significantly increases the probability of radiative transitions between the excited and ground states, thereby enhancing the fluorescence intensity. The Zn@C-210 in a solid state and Zn@C-260 in water exhibited approximately 71.50% and 21.1% quantum yields, respectively. Both Zn@C-210 and Zn@C-260 exhibited excitation-independent luminescence, featuring a long fluorescence lifetime of 6.5 μs for Zn@C-210 and 6.2 μs for Zn@C-260. Impressively, zinc-doped CDs displayed exceptional biosafety, showing no acute toxicity even at 1000 mg/kg doses. Zn@C-210 has excellent fluorescence in a solid state, showing promise in anti-photobleaching applications; meanwhile, the dual functionality of Zn@C-260 makes it useful as a folate sensor and cellular imaging probe. These findings not only advance the fundamental understanding of metal-doped carbon dot photophysics but also provide practical guidelines for developing targeted biomedical nanomaterials through rational surface engineering and doping strategies. Full article