Fluorescence Microscopy Research Articles

Environmentally friendly high-content polyurethane modified asphalt: Workability evaluation and early performance evolution

High-content polyurethane modified asphalt (HPMA) has ignited the enthusiasm of researchers due to its low-carbon characteristics and excellent performance. The workability and service performance of HPMA are both affected by complex curing reaction. The viscosity of HPMA changes over time at simulated construction temperature, and the evolution of the chemical structure and rheological properties at different environmental temperatures at the early stage were systematically investigated in this work. After thorough exploration, the construction tolerance time for HPMA-50 % (with isocyanate pre-polymer content of 50 %) and HPMA-30 % (with isocyanate pre-polymer content of 30 %) at a construction temperature of 130 ℃ are 46 minutes and 72 minutes, respectively, which can basically meet the construction time requirements. The complex modulus of HPMA gradually increases with the increasing curing time, and the phase angle temperature dependence of HPMA-50 % is significantly weakened compared to HPMA-30 %, its viscoelasticity gradually transforms to thermoset. Meanwhile, the high-temperature rutting factor improvement rate of HPMA-50 % reaches 142.1 % after curing for 14 days, indicating a tremendously improved rutting resistance. The high-temperature creep recovery rates of HPMA-50 % and HPMA-30 % increased from 65.9 % and 3.6–96.1 % and 7.6 %, respectively, after 14 days of curing. This reveals that increasing the isocyanate pre-polymer content and extending the curing time all can significantly improve the entropy-elastic properties and creep recovery ability of HPMA. Fluorescence microscopy analysis showed that the polyurethane particles in HPMA changed from a granular structure to a network structure during the curing process, and gradually transitioned to a continuous phase as the pre-polymer content increased. Further research also revealed a good correlation between the conversion rate of isocyanates and the rheological performance index of HPMA. Additionally, a functional model between the rheological performance index of HPMA and curing times was successfully established, which accurately predicted the evolution law of the rheological performance of HPMA under different ambient temperatures. The evolution mechanism of the early performance of environmentally friendly HPMA was investigated in this work, which laid the methodological foundation and theoretical basis for determining the construction tolerance time of HPMA and accurately evaluating its performance at the early stage.

Biofilm growth Dynamics in a Micro-Irrigation with Reclaimed Wastewater in the Field scale.

The dripper clogging due to the development of biofilm can reduce the benefits of micro-irrigation technology implementation using reclaimed wastewater. The narrow cross-section and labyrinth geometry of the dripper channel enhance the fouling mechanisms. The aim of this study was to evaluate the water distribution and biofouling of drip irrigation systems at the field scale during irrigation with treated wastewater. Six 100 m lines of commercial pipes with two pressure-compensating dripper types (flow rate, Q, of 0.65 L.h-1 and 1.5 L.h-1, respectively) were monitored for four months. Different zones along the pipes were selected to evaluate the influence of hydrodynamical conditions (Reynolds number = 5400 to 0) on biofouling. Destructive methods involving the biofilm extraction by mechanical means, showed little biofilm development without significant differences in dry and organic matter content in function of the sampling location along the pipe or dripper flow rate (Q0.65 and Q1.5). These results were confirmed by non-destructive methods, such as optical coherence tomography, that nevertheless showed that biofouling concerned 15-20% of the total dripper labyrinth volume. Total organic carbon monitoring and its composition (by three-dimensional excitation and emission matrix fluorescence microscopy) showed that the biofilm did not significantly influence the organic matter nature. Our results indicated that the biological activity and biofilm development in irrigation systems were more affected by the environmental conditions, particularly water temperature, rather than flow conditions. This confirmed that treated wastewater with low organic content can be used in micro-irrigation systems without significant loss of efficiency, even in conditions requiring intensive irrigation, such as the Mediterranean climate.

Light on abnormal red blood cell subpopulations: Label-free optics-based approach for studying in vitro rigidified blood cells

RBC deformability plays a crucial role in maintaining proper blood flow and oxygen delivery throughout the body. Conventional ektacytometry fails to differentiate between variations in RBC subpopulation deformability as the averaging measurement process obscures these differences. In this study, we introduced an approach that integrates label-free optics-based techniques (flow cytometry, phase-contrast, and two-photon excitation fluorescent microscopy) with ektacytometry to evaluate subpopulations that exhibit decreased RBC deformability upon an in vitro oxidation using 0.5 mM TBHP, as a low-level oxidative agent.We found that flow cytometry can easily detect rigidified and oxidized subpopulations based on FSC/SSC light distribution, as well as RBC fluorescence intensity and peak area likely originating from hemoglobin photo and/or degradation products. Two-photon excitation fluorescence microscopy proved altered morphology and spatial location of fluorescence intensity signal near the membrane of oxidized RBCs, when compared to control RBC, indicating a link with the reduced deformability. The proposed label-free optics-based methodology, which combines established techniques with more sophisticated microscopy, emerges as a promising tool for detecting mechano-biological changes in different RBC subpopulations induced by oxidative stress. The findings suggest potential applications in clinical practice for monitoring pathological conditions influenced by physical or environmental stress and as a quality control measure for stored RBCs.

Effect of Bioplastic Material on Adhesion, Growth, and Proliferative Activity of Human Fibroblasts When Incubated in Solutions Mimic the Acidity of Wound an Acute and Chronic Inflammation.

: The aim of this study was to characterize the effect of bioplastic material based on collagen, hyaluronic acid, and elastin on the viability and proliferative activity of human fibroblasts in conditions simulating the acidity of acute and chronic wounds. : Bioplastic material was made according to the method described in RF patent no.2722744. Adhesive properties and proliferative activity of human fibroblasts were assessed visually using fluorescent microscopy. The number of apoptotic and necrotic cells was assessed by flow cytometry (BD FACSCanto II) using the commercial FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). The strength, Young's modulus, and elasticity of the gels were determined on a TA.XT-plus texture analyzer (Stable Micro Systems, Great Britain). : Using the methods of luminescent microscopy and flow cytometry, we found that the cell viability (namely, adhesive properties and proliferative activity) decreases after incubation on condition mimic of physiological acidosis. We found that bioplastic material contributes to the preservation of adhesive properties, viability, and proliferative activity of fibroblasts in physiological acidosis conditions. The results obtained indicate that bioplastic material based on soluble dermis components can be used as a biologically active component of wound dressings to increase the effectiveness of reparative regeneration, especially in cases of excessive acute inflammation.

Grape pomace high-methoxyl pectin: A new prebiotic stabilizer for low-fat synbiotic yogurt gels – Optimization and characterization

High-methoxyl pectin (HMP, 72.5 % esterification degree and galacturonic acid content of 67.9 %) was extracted from grape pomace using a sequential ultrasound-microwave extraction. The extracted HMP was used to develop low-fat synbiotic set yogurts containing probiotic cells. Higher grape pomace pectin (GPP) concentrations (0.5–2 %) increased the probiotic bacterial population of Lactobacillus acidophilus LA-5 and Bifidobacterium bifidum BB-12. Higher cell viability was observed for L. acidophilus LA-5 compared to B. bifidum BB-12. A response surface optimization showed that the presence of 8.08 Log CFU mL−1L. acidophilus LA-5 and 1.88 % HMP experimentally resulted in the best probiotic viability (10.83 ± 0.11 Log CFU mL−1), overall acceptability (8.03 ± 0.06), and pH (4.25 ± 0.05) values. Compared to pectin-free probiotic yogurts, the optimal yogurt gels presented higher probiotic survivability, lower syneresis, and superior storage-dependent sensory attributes during 21 days of storage. However, a 14-day storage period was generally deemed suitable. The GPP-containing yogurt compared to the pectin-free sample exhibited higher colloidal stability with a larger particle size (433.8 nm vs. 272.5 nm) and lower zeta potential (−20.4 mV vs. −10.6 mV). Field emission-scanning electron (FE-SEM) and fluorescent (FLM) microscopy images confirmed a denser microstructure for GPP-enriched yogurts. The chemical interactions in the yogurt were not affected by enriching with GPP as investigated by FTIR, whereas the steady and dynamic rheological properties were significantly improved. GPP-enriched yogurt had a firmer gel structure with a larger linear region and lower G′ compared to the control, indicating a semi-solid state. The GPP as a multi-functional prebiotic ingredient would be promising in designing healthier food products.

Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin

Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin.

Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.

BackgroundBabesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.MethodsThe endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).ResultsIn this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.ConclusionsThis study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.Graphical

Fluorescent Microscopy Investigation of Cytotoxic Impact on MCF-7 Cell Line Treated With Zinc Nanoparticles Synthesized From Jania rubens.

Zinc nanoparticles (ZnNPs) are a viable option in a number of disciplines, including cancer treatment, due to their special features. Among the several techniques for synthesizing ZnNP, biosynthesis with natural extracts is a highly effective and environmentally benign method, especially for uses in biomedicine. Using an aqueous extract of the marine red seaweed Jania rubens, we created a unique biosynthetic technique in this study to manufacture ZnNPs. The produced ZnNPs have a characteristic flower-like form, as seen by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effective production of ZnNPs and the involvement of biomolecules in the synthesis process were validated by energy-dispersive X-ray spectroscopy (EDAX) and Fourier transform infrared spectroscopy (FTIR) techniques. Using the MTT assay, the cytotoxic effects of the biosynthesized ZnNPs were evaluated, indicating their ability to inhibit MCF-7 breast cancer cells. Furthermore, ZnNPs' cytotoxicity against MCF-7 cells was validated by live/dead imaging experiments, which supported the MTT results.

Fine-tuned spatiotemporal dynamics of DNA replication during phage lambda infection.

Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.

Simultaneous dual-colour labelling of mitochondria and lysosomes: An indolium-based approach

Fluorescence is a valuable tool for understanding mitochondria-lysosome interactions, which is central for unveiling their roles in health and disease. Herein, we describe novel indolium-based molecules for labelling mitochondria and lysosomes simultaneously with organelle-specific fluorescence properties. These probes readily enable clear distinction of mitochondria and lysosomes by combining confocal and two-photon fluorescence microscopy. Our results show that this unique behaviour relies on aggregation. The functioning of these probes is not compromised by mitophagy, allowing the visualisation of these organelles in separate emission channels by using a single fluorescent marker under all scenarios. Their enhanced experimental simplicity compared to using multiple dyes makes these novel compounds well-suited for automated screening applications within living cells.

Lactic Acid Bacteria Surface Proteins in the Mechanisms of Cell Adhesion and Immunoregulation

ABSTRACTThis study delves into the role of lactic acid bacteria (LAB) surface proteins in cell adhesion and immunoregulation. Using fluorescence microscopy, we observed distinct adhesion patterns on various cell types. LAB surface proteins demonstrated concentration‐dependent inhibition of Salmonella adhesion, with LAB69 exhibiting potent antagonistic effects. Genetic expression analysis revealed nuanced responses in key genes (MD2, TLR4, IL‐10, MUC3, MIF) across different cell types, highlighting the diverse immunomodulatory effects of LAB surface proteins. Modulation of pro‐inflammatory (TNF‐α) and anti‐inflammatory (IL‐10) cytokines further emphasized the complex interplay. In conclusion, this study underscores the pivotal role of LAB surface proteins in mediating cell adhesion and immunoregulation, providing a foundation for isolating specific immunomodulatory molecules within LAB surface proteins for potential applications in microbial ecological agents.

Label-free rat brain traumatic penumbra imaging based on multiphoton fluorescence microscopy

Label-free rat brain traumatic penumbra imaging based on multiphoton fluorescence microscopy

Investigating antibacterial and anti-inflammatory properties of synthetic curcuminoids

The concept of intratumoral microbiota is gaining attention in current research. Tumor-associated microbiota can activate oncogenic signaling pathways such as NF-κB, thereby promoting tumor development and progression. Numerous studies have demonstrated that curcumin and its analogs possess strong antitumor effects by targeting the NF-κB signaling pathway, along with potent antibacterial properties. In this study, we tested the antibacterial activity of two curcuminoids, Py-cPen and V-cPen, against the Gram-negative bacterial strains Pseudomonas aeruginosa and Escherichia coli and the Gram-positive bacterial strain Streptococcus aureus using in vitro assays and fluorescent microscopy. We observed that both Py-cPen and V-cPen reduced NF-κB activation upon lipopolysacharide (LPS) challenge in cell assays. In addition, our findings indicate that Py-cPen and V-cPen interact with LPS, as demonstrated by transmission electron microscopy and confirmed using in silico analyses, thereby modulating LPS activity. Overall, our data indicate that Py-cPen and V-cPen exhibit strong antibacterial and antiinflammatory properties, suggesting their potential as candidates for new multitarget therapeutic strategies.

Evaluation of biocompatibility and osteoconductivity of a hybrid cell-tissue graft for bone regenerative medicine

Aim – to evaluate in vitro the biocompatibility and osteoconductivity of a hybrid graft based on a bioorganic matrix, human bone marrow mesenchymal stromal cells (BM-MSC) and osteogenic growth factors. Material and methods. Bioorganic matrices were studied for biocompatibility with human BM-MSC culture used in traumatology and orthopedics. For promoted osteogenic differentiation of BM-MSCs, allogeneic plasma enriched with soluble platelet factors was used. The osteogenic potential of BM-MSCs by the synthesis of mRNAs of early (transcription factor 2 (Run X2), alkaline phosphatase (ALP)) and late genes (osteopontin (OSP)) osteogenesis was analyzed. The properties of cell adhesion and proliferation of MSCs in the conditions of a three-dimensional hybrid graft by the MTT test and fluorescence microscopy were assessed. Results. The biocompatibility of the studied bioorganic matrices with human BM-MSCs was established. The collagen matrix promoted rapid cell adhesion and proliferation between the scaffold fibrils. It has also been established that allogeneic platelet-rich plasma affects the osteogenic differentiation of human BM-MSCs in vitro, increasing the expression of marker genes RunX2, ALP, OSP. When modeling a hybrid graft in vitro, the formation of a tight contact between the alloimplant and collagen biopolymer using MSCs was shown. Conclusion. The biological properties of the developed hybrid cell-tissue graft characterize its biocompatibility and osteoconductivity of its constituent components, which makes it promising for use in regenerative medicine, especially in reconstructive surgery of bone defects.

Impact of Probiotic Fermentation on the Physicochemical Properties of Hemp Seed Protein Gels

Hemp seed protein isolates (HPI) were used to produce a gel through probiotic fermentation. This study assessed how fermentation time (ranging from 0 to 16 h) affected the physicochemical properties of the HPI gel. The results indicated that gel formation began after 8 h of fermentation, as demonstrated by a pH decrease, an increase in particle size, and the development of aggregation observed through fluorescence and scanning electron microscopy. The gel produced after 16 h of fermentation showed the highest viscosity, storage modulus, and gel strength, attributed to stronger molecular interactions, including non-covalent and covalent crosslinking. However, the gel produced after 12 h of fermentation showed the highest water-holding capacity, and extending the fermentation beyond 12 h caused a decrease in water-holding capacity. Additionally, the subunits tended to form polymers after fermentation, suggesting that gel formation was influenced by both acidification and specific covalent crosslinking. These findings propose that HPI could serve as a viable alternative for developing plant-based gel products.

Electric Demulsification Membrane Technology for Confined Separation of Oil-Water Emulsions.

Demulsification technology for separation of oil-water (O/W) emulsions, especially those stabilized by surfactants, is urgently needed yet remains highly challenging due to their inherent stability characteristics. Electrocoalescence has emerged as a promising solution owing to its simplicity, efficacy, and versatility, yet hindered by substantial energy consumption (e.g., >50 kWh/m3) along with undesirable Faradic reactions. Herein, we propose an innovative electric demulsification technology that leverages conductive membrane microchannels to confine oil droplets from the oil-water emulsion for achieving high energy-efficient coalescence of oil droplets. The proposed system reduces the required voltage down to 12 V, 2 orders of magnitude lower than that of conventional electrocoalescence systems, while achieving a similar separation efficacy of 91.4 ± 3.0% at a low energy consumption (3 kWh/m3) and an ultrahigh permeability >3000 L/(m2·h·bar). In situ fluorescence microscopy combined with COMSOL simulations provided insight into the fundamental mechanistic steps of an electric demulsification process confined to membrane microchannels: (1) rapid electric-field redistribution of oil droplet surfactant molecules, (2) enhanced collision probability due to confined oil droplet concentration under dielectrophoretic forces, and (3) increased collision efficacy facilitated by the membrane pore structure. This strategy may revolutionize the next generation of demulsification and oil-water separation innovations.

A programmable platform for probing cell migration and proliferation.

The advent of advanced robotic platforms and workflow automation tools has revolutionized the landscape of biological research, offering unprecedented levels of precision, reproducibility, and versatility in experimental design. In this work, we present an automated and modular workflow for exploring cell behavior in two-dimensional culture systems. By integrating the BioAssemblyBot® (BAB) robotic platform and the BioApps™ workflow automater with live-cell fluorescence microscopy, our workflow facilitates execution and analysis of in vitro migration and proliferation assays. Robotic assistance and automation allow for the precise and reproducible creation of highly customizable cell-free zones (CFZs), or wounds, in cell monolayers and "hands-free," schedulable integration with real-time monitoring systems for cellular dynamics. CFZs are designed as computer-aided design models and recreated in confluent cell layers by the BAB 3D-Bioprinting tool. The dynamics of migration and proliferation are evaluated in individual cells using live-cell fluorescence microscopy and an in-house pipeline for image processing and single-cell tracking. Our robotics-assisted approach outperforms manual scratch assays with enhanced reproducibility, adaptability, and precision. The incorporation of automation further facilitates increased flexibility in wound geometry and allows for many experimental conditions to be analyzed in parallel. Unlike traditional cell migration assays, our workflow offers an adjustable platform that can be tailored to a wide range of applications with high-throughput capability. The key features of this system, including its scalability, versatility, and the ability to maintain a high degree of experimental control, position it as a valuable tool for researchers across various disciplines.

Retracted] Activating autophagy and ferroptosis of 3‑Chloropropane‑1,2‑diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the flow cytometric assay data shown in Fig. 2C on p. 5, certain of the fluorescence microscopy data shown in Fig. 4B on p. 7, the FerroOrange‑stained cellular data in Fig. 5B and the C11‑BODIPY581/591‑stained cellular data in Fig. 5C on p. 8, and the cell autophagic data in Fig. 6E and G on p. 9 were strikingly similar to data that had already been submitted for publication in different form in different articles written by different authors at different research institutes (a few of which have now been retracted). Owing to the fact that the contentious data in the above article had already been published, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 27: 76, 2023; DOI: 10.3892/mmr.2023.12963].

Mechanisms of atrial-fibrillation-induced ventricular dysfunction and recovery in the human left ventricular myocardium

Abstract Background Atrial fibrillation (AF) frequently occurs alongside heart failure (HF). Clinical studies indicate improved left ventricular (LV) function following rhythm restoration but the underlying myocardial processes remain unclear. Purpose This study investigated effects of AF and its termination on human LV myocardium as well as potential molecular mechanisms involved in the AF-induced LV dysfunction. Methods Human LV tissue slices obtained from HF patients were long-term cultivated in-vitro. AF was simulated by tachy-arrhythmic culture pacing at 100 bpm with 30% irregularity for 7 days and contractile LV function was compared to slices undergoing sinus rhythm (SR)-simulation (60 bpm/0%). After 7 days, rhythm restoration was simulated by switching to SR-simulation. To elucidate cellular mechanisms involved in AF-induced LV dysfunction, human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were subjected to AF-simulation and examined via epifluorescence microscopy (FURA-2) and confocal microscopy (Fluo-4). Additionally, to investigate the impact of reactive oxygen species (ROS) on cardiomyocytes, iPSC-CM were subjected to treatment with the ROS-scavenger N-acetylcysteine (NAC). Results AF-simulation caused a progressive decline in systolic force of human LV myocardium (n=14 slices) with significantly reduced twitch amplitudes compared to SR-simulation (n=11). Within just 5 days of AF-termination, a significant recovery in LV function was observed, indicated by improved systolic contraction amplitudes. IPSC-CM measurements revealed a significant reduction in systolic Ca2+-transient amplitudes as well as an increased diastolic Ca2+ leak after AF-simulation (n=41) compared to SR-simulation (n=44). However, 5 days after SR-restoration, iPSC-CM exhibited no significant differences anymore, indicating a fast recovery of systolic Ca2+-cycling. We further investigated the impact of ROS on LV function and could show that Ca2+-transient amplitude was preserved after AF-simulation in iPSC-CM when treated with NAC (n=35), suggesting an interplay of ROS with the Ca2+-homeostasis of cardiomyocytes. Conclusions This study demonstrates that AF impairs LV function in human HF myocardium. However, the involved changes in contractility and cardiomyocyte Ca2+ handling are reversible upon AF termination leading to improved LV function already within few days after AF cessation. Moreover, we provided an indication for an interplay of ROS with the Ca2+ homeostasis, endorsing an AF-induced LV-dysfunction, which merits further investigation.