Microplastics in Asian rivers: Geographical distribution, most detected types, and inconsistency in methodologies.

Microplastics pose a significant environmental threat, with potential implications for toxic chemical release, aquatic life endangerment, and human food chain contamination. In Asia, rapid economic growth coupled with inadequate waste management has escalated plastic pollution in rivers, positioning them as focal points for environmental concern. Despite Asia's rivers being considered the most polluted with plastics globally, scholarly attention to microplastics in the region's freshwater environments is a recent development. This study undertakes a systematic review of 228 scholarly articles to map microplastic hotspots in Asian freshwater systems and synthesize current research trends within the continent. Findings reveal a concentration of research in China and Japan, primarily investigating riverine and surface waters through net-based sampling methods. Polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET) emerge as the predominant microplastic types, frequently observed as fibers or fragments. However, the diversity of sampling methodologies and reporting metrics complicates data synthesis, underscoring the need for standardized analytical frameworks to facilitate comparative analysis. This paper delineates the distribution of microplastic hotspots and outlines the prevailing challenges and prospects in microplastic research within Asian freshwater contexts.

More than double the fun with two-photon excitation microscopy.

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies.

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging.

Fluorescent biosensors revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells, yet the current technology is limited in resolution and dimensionality. Here, we introduce highly sensitive chemigenetic kinase activity biosensors that combine the genetically encodable self-labeling protein tag HaloTag7 with bright far-red-emitting synthetic fluorophores. This technology enables five-color biosensor multiplexing, 4D activity imaging, and functional super-resolution imaging via stimulated emission depletion (STED) microscopy.

Investigating Diffusion Dynamics and Interactions with Scanning Fluorescence Correlation Spectroscopy (sFCS).

Activation of immune cells and formation of immunological synapses (IS) rely critically on the reorganization of the plasma membrane. These highly orchestrated processes are driven by diffusion and oligomerization dynamics, as well as by single molecule interactions. While slow macro- and meso-scale changes in organization can be observed with conventional imaging, fast nano-scale dynamics are often missed with traditional approaches, but resolving them is, nonetheless, essential to understand the underlying biological mechanisms at play. Here, we describe the use of scanning fluorescence correlation spectroscopy (sFCS) and scanning fluorescence cross-correlation spectroscopy (sFCCS) to study reorganization and changes in molecular diffusion dynamics and interactions during IS formation and in other biological settings. We focus on the practical aspects of the measurements including calibration and alignment of the optical setup, present a comprehensive protocol to perform the measurements, and provide data analysis pipelines and strategies. Finally, we show an exemplary application of the technology to studying Lck diffusion during T-cell signaling.

Material flow analysis of commercial fishing gears in Taiwan.

Marine debris is an international environmental issue, and the growing amount of abandoned, lost, or otherwise discarded fishing gear (ALDFG) is a particular concern. Despite Taiwan's substantial fishing industry, there is a lack of comprehensive understanding of fishing gear. This work conducted a static material flow analysis to estimate the flows and the stocks of fishing gear in Taiwan in 2020, based on government statistics and interviews with fishing gears producing companies, fishermen, and recycling companies. Our findings reveal that the inflow, outflow, and stock of the fishing gears are 8,846 t/a, 4,271 t/a, and 4,575 t/a, respectively. Only 36 % of end-of-life fishing gear is recycled, while the rest is incinerated or landfilled. Additionally, the stock comprises 27 % in use, 23 % in ports, and 50 % entering the ocean. These results underscore the need to increase recycling capacity, prevent loss in oceans, and promote repairs to extend the lifespan of fishing gear.

Evaluating the waste and CO2 reduction potential of packaging by reuse model in supermarkets in Taiwan.

Consumption of single-use packaging has been increasing globally and the waste produced causes negative impacts on both human and the environment. Retailers, such as supermarkets, developed quickly in recent years to provide for the modern lifestyle, using a lot of packaging in the process of distribution and sales. This research evaluates the packaging waste and CO2 reduction potential of 10 different products sold in supermarkets in Taiwan when adopting different reuse strategies of Reduce, Return and Refill. In the suggested reuse strategies, a total of 8 kilotons of packaging waste and 30 kilotons of packaging CO2 can be reduced, accounting for 50.8% and 59.8% reduction of the current situation, respectively. Retailers are suggested to provide different reuse strategies and experiential activities to increase consumers familiarity with new consumption methods. Significant impacts are made with a slight change in the small proportion investigated, which suggests considerable benefits if the scope is expanded.

Microplastics in rivers along an urban-rural gradient in an urban agglomeration: Correlation with land use, potential sources and pathways.

Microplastics are ubiquitous and affect all environments, including rivers. In recent years the number of studies about microplastics in rivers has strongly increased. But still many questions exist regarding sources, pathways, and the role of land use patterns. In this study the relationship between microplastics abundance and anthropogenic factors (population density, urbanization, land use types), as well as the potential role of storm sewers as pathways in tributaries of the Wu River in Taichung, central Taiwan, were studied. Two river catchments of the Dali River were studied in greater detail to investigate the influence of land use on microplastics abundance along an urban-rural gradient, and to observe the change of microplastics abundance in the transition from rural to urban areas. Samples were taken from 41 different locations in urban and rural areas using a manta net with a mesh size of 0.3 mm. Results show abundances ranging from 0 pcs/m³ in unpopulated rural areas up to 230 pcs/m³ in densely populated urban centers, and are positively correlated with population density. Remarkably, a sharp increase in microplastics abundance was observed at the transition from rural to urban areas, which coincides with the appearance of storm sewers. Land use analysis revealed that microplastics abundance positively correlates with the size of industrial, residential and traffic areas in the catchment areas, and negatively correlates with the size of forest areas. Source areas for microplastics in the studied rivers are likely residential and commercial areas. Furthermore, the results of this study show that correlations between microplastics abundances and population density or land use patterns along urban-rural gradients are not trivial. Strength of correlations can depend on local factors or how well urban-rural gradients are developed. Absence of correlations need to be considered carefully, as existing correlations might be masked by the above-mentioned factors.

The kinase occupancy of T cell coreceptors reconsidered.

The sensitivity of the αß T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αß, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αß/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to also show that stoichiometric coupling to Lck required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 outcompeted CD8αß for Lck. In T cells, TCR signaling induced CD4/Lck oligomerization but did not affect the high levels of CD4/Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.

ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis.

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.

Material studies for the recycling of abandoned, lost or otherwise discarded fishing gear (ALDFG).

This study investigates the feasibility of material recycling for retrieved gillnets from the Baltic Sea collected during a campaign of the World Wildlife Fund (WWF) Germany. Fragments from the material were analysed by Fourier transform infrared (FTIR) spectroscopy revealing polyamide 6 (PA6), polypropylene (PP) and polyethylene terephthalate (PET) in net material, swim lines and sink lines, respectively. A visual examination by microscope found large quantities of minerals attached to the surface of the material as well as in knots and loops of the polymer structure. Ash tests showed that a pre-treatment of the material including sorting, shredding, density separation and washing allows to reduce the mineral content from more than 45% of the total to 1.1%. However, for a separation by density, it is important that the entangled fibres can move freely. This is a major challenge for a primary or secondary mechanical recycling because a substantial fibre length reduction is required for the small polymer fibres down to a diameter of 20 µm. Another challenge for all kinds of recycling is the presence of lead lines in gillnets. Automated technology for removing these does not exist until now. A manual removal is indispensable to limit the level of contamination. Due to the complex pre-treatment and the elevated heavy metal concentrations also a tertiary or feedstock recycling seems not to be a possible pathway for retrieved gillnets. Yet, other options such as a primary recycling in concrete or bitumen additives or quaternary recycling via incineration may be conceivable alternatives. But there are also some arguments against these options. Better product design must be the goal to prevent plastic pollution and establish a functioning circular economy. In this context, the heavy metal contamination by abandoned, lost or otherwise discarded fishing gear (ALDFG) must be stopped.

Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells.

To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.

Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins.

Advanced fluorescence microscopy studies require specific and monovalent molecular labeling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labeled nanobodies against commonly employed fluorescent proteins (FPs). However, very little is known how these nanobodies influence their target molecules. Here, we tested commercially available nanobodies and observed clear changes of the fluorescence properties, mobility and organization of green fluorescent protein (GFP) tagged proteins after labeling with the anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently labeled nanobodies with the GFP-labeled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, likely oligomerized, form of the FPs, promoting disassembly into monomeric form after binding. Our findings have significant implications on the application of nanobodies and GFP labeling for studying dynamic and quantitative protein organization in the plasma membrane of living cells using advanced imaging techniques.

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy.

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1.

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Quantitative Bio-Imaging Tools to Dissect the Interplay of Membrane and Cytoskeletal Actin Dynamics in Immune Cells.

Cellular function is reliant on the dynamic interplay between the plasma membrane and the actin cytoskeleton. This critical relationship is of particular importance in immune cells, where both the cytoskeleton and the plasma membrane work in concert to organize and potentiate immune signaling events. Despite their importance, there remains a critical gap in understanding how these respective dynamics are coupled, and how this coupling in turn may influence immune cell function from the bottom up. In this review, we highlight recent optical technologies that could provide strategies to investigate the simultaneous dynamics of both the cytoskeleton and membrane as well as their interplay, focusing on current and future applications in immune cells. We provide a guide of the spatio-temporal scale of each technique as well as highlighting novel probes and labels that have the potential to provide insights into membrane and cytoskeletal dynamics. The quantitative biophysical tools presented here provide a new and exciting route to uncover the relationship between plasma membrane and cytoskeletal dynamics that underlies immune cell function.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

Nanoscale dynamics of cholesterol in the cell membrane.

Cholesterol constitutes ∼30-40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.

Measuring nanoscale diffusion dynamics in cellular membranes with super-resolution STED-FCS.

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.

Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer.

Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane's disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.