Current Progress in Expansion Microscopy: Chemical Strategies and Applications.

Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.

Identification of prognostic biomarkers for cholangiocarcinoma by combined analysis of molecular characteristics of clinical MVI subtypes and molecular subtypes.

Cholangiocarcinoma (CCA) is widely noted for its high degree of malignancy, rapid progression, and limited therapeutic options. This study was carried out on transcriptome data of 417 CCA samples from different anatomical locations. The effects of lipid metabolism related genes and immune related genes as CCA classifiers were compared. Key genes were derived from MVI subtypes and better molecular subtypes. Pathways such as epithelial mesenchymal transition (EMT) and cell cycle were significantly activated in MVI-positive group. CCA patients were classified into three (four) subtypes based on lipid metabolism (immune) related genes, with better prognosis observed in lipid metabolism-C1, immune-C2, and immune-C4. IPTW analysis found that the prognosis of lipid metabolism-C1 was significantly better than that of lipid metabolism-C2 + C3 before and after correction. KRT16 was finally selected as the key gene. And knockdown of KRT16 inhibited proliferation, migration and invasion of CCA cells.

Insights into the pH effect on hydrogen electrocatalysis.

Hydrogen electrocatalytic reactions, including the hydrogen evolution reaction (HER) and the hydrogen oxidation reaction (HOR), play a crucial role in a wide range of energy conversion and storage technologies. However, the HER and HOR display anomalous non-Nernstian pH dependent kinetics, showing two to three orders of magnitude sluggish kinetics in alkaline media compared to that in acidic media. Fundamental understanding of the origins of the intrinsic pH effect has attracted substantial interest from the electrocatalysis community. More critically, a fundamental molecular level understanding of this effect is still debatable, but is essential for developing active, stable, and affordable fuel cells and water electrolysis technologies. Against this backdrop, in this review, we provide a comprehensive overview of the intrinsic pH effect on hydrogen electrocatalysis, covering the experimental observations, underlying principles, and strategies for catalyst design. We discuss the strengths and shortcomings of various activity descriptors, including hydrogen binding energy (HBE) theory, bifunctional theory, potential of zero free charge (pzfc) theory, 2B theory and other theories, across different electrolytes and catalyst surfaces, and outline their interrelations where possible. Additionally, we highlight the design principles and research progress in improving the alkaline HER/HOR kinetics by catalyst design and electrolyte optimization employing the aforementioned theories. Finally, the remaining controversies about the pH effects on HER/HOR kinetics as well as the challenges and possible research directions in this field are also put forward. This review aims to provide researchers with a comprehensive understanding of the intrinsic pH effect and inspire the development of more cost-effective and durable alkaline water electrolyzers (AWEs) and anion exchange membrane fuel cells (AMFCs) for a sustainable energy future.

Modulating the RPS27A/PSMD12/NF-κB pathway to control immune response in mouse brain ischemia-reperfusion injury.

BACKGROUND: Investigating immune cell infiltration in the brain post-ischemia-reperfusion (I/R) injury is crucial for understanding and managing the resultant inflammatory responses. This study aims to unravel the role of the RPS27A-mediated PSMD12/NF-κB axis in controlling immune cell infiltration in the context of cerebral I/R injury. METHODS: To identify genes associated with cerebral I/R injury, high-throughput sequencing was employed. The potential downstream genes were further analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses. For experimental models, primary microglia and neurons were extracted from the cortical tissues of mouse brains. An in vitro cerebral I/R injury model was established in microglia using the oxygen-glucose deprivation/reoxygenation (OGD/R) technique. In vivo models involved inducing cerebral I/R injury in mice through the middle cerebral artery occlusion (MCAO) method. These models were used to assess neurological function, immune cell infiltration, and inflammatory factor release. RESULTS: The study identified RPS27A as a key player in cerebral I/R injury, with PSMD12 likely acting as its downstream regulator. Silencing RPS27A in OGD/R-induced microglia decreased the release of inflammatory factors and reduced neuron apoptosis. Additionally, RPS27A silencing in cerebral cortex tissues mediated the PSMD12/NF-κB axis, resulting in decreased inflammatory factor release, reduced neutrophil infiltration, and improved cerebral injury outcomes in I/R-injured mice. CONCLUSION: RPS27A regulates the expression of the PSMD12/NF-κB signaling axis, leading to the induction of inflammatory factors in microglial cells, promoting immune cell infiltration in brain tissue, and exacerbating brain damage in I/R mice. This study introduces novel insights and theoretical foundations for the treatment of nerve damage caused by I/R, suggesting that targeting the RPS27A and downstream PSMD12/NF-κB signaling axis for drug development could represent a new direction in I/R therapy.

FGCNSurv: dually fused graph convolutional network for multi-omics survival prediction.

MOTIVATION: Survival analysis is an important tool for modeling time-to-event data, e.g. to predict the survival time of patient after a cancer diagnosis or a certain treatment. While deep neural networks work well in standard prediction tasks, it is still unclear how to best utilize these deep models in survival analysis due to the difficulty of modeling right censored data, especially for multi-omics data. Although existing methods have shown the advantage of multi-omics integration in survival prediction, it remains challenging to extract complementary information from different omics and improve the prediction accuracy. RESULTS: In this work, we propose a novel multi-omics deep survival prediction approach by dually fused graph convolutional network (GCN) named FGCNSurv. Our FGCNSurv is a complete generative model from multi-omics data to survival outcome of patients, including feature fusion by a factorized bilinear model, graph fusion of multiple graphs, higher-level feature extraction by GCN and survival prediction by a Cox proportional hazard model. The factorized bilinear model enables to capture cross-omics features and quantify complex relations from multi-omics data. By fusing single-omics features and the cross-omics features, and simultaneously fusing multiple graphs from different omics, GCN with the generated dually fused graph could capture higher-level features for computing the survival loss in the Cox-PH model. Comprehensive experimental results on real-world datasets with gene expression and microRNA expression data show that the proposed FGCNSurv method outperforms existing survival prediction methods, and imply its ability to extract complementary information for survival prediction from multi-omics data. AVAILABILITY AND IMPLEMENTATION: The codes are freely available at https://github.com/LiminLi-xjtu/FGCNSurv.

Single-molecule tracking technology based on symmetric power exponential phases.

We present a design approach for a symmetric power exponential phase, resulting in a phase-modulated point-spread function with two symmetrical mainlobes that can be steered toward the optical axis opposite the x axis. This design offers nanoscale 3D localization capabilities suitable for 3D single-molecule tracking and localization imaging. The axial probing depth of this symmetric power exponential point-spread function can be adjusted as needed by manipulating specific parameters. Optimization of the symmetric power exponential phase involves truncation filtering to reduce sidelobes and the utilization of a phase inversion-based optimization algorithm to enhance transfer function efficiency and localization precision. A successful multi-molecule 3D tracking experiment was conducted at a 10 µm axial depth using a numerical aperture of 1.4 to validate the efficacy of the proposed design methodology.

Anxiolytic effect of antidiabetic metformin is mediated by AMPK activation in mPFC inhibitory neurons.

Diabetic patients receiving the antidiabetic drug metformin have been observed to exhibit a lower prevalence of anxiety disorders, yet the precise mechanism behind this phenomenon is unclear. In our study, we found that anxiety induces a region-specific reduction in AMPK activity in the medial prefrontal cortex (mPFC). Concurrently, transgenic mice with brain-specific AMPK knockout displayed abnormal anxiety-like behaviors. Treatment with metformin or the overexpression of AMPK restored normal AMPK activity in the mPFC and mitigated social stress-induced anxiety-like behaviors. Furthermore, the specific genetic deletion of AMPK in the mPFC not only instigated anxiety in mice but also nullified the anxiolytic effects of metformin. Brain slice recordings revealed that GABAergic excitation and the resulting inhibitory inputs to mPFC pyramidal neurons were selectively diminished in stressed mice. This reduction led to an excitation-inhibition imbalance, which was effectively reversed by metformin treatment or AMPK overexpression. Moreover, the genetic deletion of AMPK in the mPFC resulted in a similar defect in GABAergic inhibitory transmission and a consequent hypo-inhibition of mPFC pyramidal neurons. We also generated a mouse model with AMPK knockout specific to GABAergic neurons. The anxiety-like behaviors in this transgenic mouse demonstrated the unique role of AMPK in the GABAergic system in relation to anxiety. Therefore, our findings suggest that the activation of AMPK in mPFC inhibitory neurons underlies the anxiolytic effects of metformin, highlighting the potential of this primary antidiabetic drug as a therapeutic option for treating anxiety disorders.

Water-enabling strategies for asymmetric catalysis.

Water possesses unique advantages, including abundance, environmental friendliness and mild effects. Undoubtedly, it is an ideal solvent or reagent in chemical syntheses. Water also shows unique abilities in catalytic asymmetric synthesis. It can accelerate reaction rates, improve diastereo- or enantioselectivities, initiate reactions, diversify chemo, diastereo- or enantioselectivities through various effects (hydrophobic, hydrogen bonding, protonation). Several reviews have demonstrated the positive effects of water in asymmetric synthesis. In this review, we summarize water-enabling strategies in the last decade, and focus on advances which reveal how water affects a reaction.

Tofacitinib prevents depressive-like behaviors through decreased hippocampal microgliosis and increased BDNF levels in both LPS-induced and CSDS-induced mice.

Depressive disorders are a global mental health challenge that is closely linked to inflammation, especially in the post-COVID-19 era. The JAK-STAT pathway, which is primarily associated with inflammatory responses, is not fully characterized in the context of depressive disorders. Recently, a phase 3 retrospective cohort analysis heightened that the marketed JAK inhibitor tofacitinib is beyond immune diseases and has potential for preventing mood disorders. Inspired by these clinical facts, we investigated the role of the JAK-STAT signaling pathway in depression and comprehensively assessed the antidepressant effect of tofacitinib. We found that aberrant activation of the JAK-STAT pathway is highly conserved in the hippocampus of classical depressive mouse models: LPS-induced and chronic social defeat stress (CSDS)-induced depressive mice. Mechanistically, the JAK-STAT pathway mediates proinflammatory cytokine production and microgliosis, leading to synaptic defects in the hippocampus of both depressive models. Remarkably, the JAK inhibitor tofacitinib effectively reverses these phenomena, contributing to its antidepressant effect. These findings indicate that the JAK/STAT pathway could be implicated in depressive disorders, and suggest that the JAK inhibitor tofacitinib has a potential translational implication for preventing mood disorders far beyond its current indications.

Response of the algal-bacterial community to thermal stratification succession in a deep-water reservoir: Community structure, co-assembly patterns, and functional groups.

Thermal stratification in lakes and reservoirs may intensify and become more persistent with global warming. Periodic thermal stratification is a naturally occurring phenomenon that indicates a transition in aquatic ecosystem homeostasis, which could lead to the deterioration of water quality and impaired aquatic communities. However, the responses of communities and associated nutrient cycling processes to periodic thermal stratification are still poorly understood. This study delved into the changes in water quality, algal-bacterial communities, and functional diversity influenced by thermal stratification succession, and their relationship with nutrient cycling. The results indicated that the apparent community dynamics were driven by environmental factors, with ammonium (NH4+) and nitrate (NO3--N) being the most important factors that influenced the algal and bacterial community structure, respectively. Ecological niche widths were narrower during thermal stratification, exacerbating the antagonism of the communities, and stochastic processes dominated community assembly. Then, the complexities of the co-occurrence network decreased with succession. Algal community assembly became more deterministic, while bacterial assembly became more stochastic. Moreover, the roles of algal-bacterial multidiversity in nutrient cycling differed: bacterial diversity enhanced nutrient cycling, whereas algal diversity had the opposite effect. These findings broadened our understanding of microbial ecological mechanisms to environmental change and provided valuable ecological knowledge for securing water supplies in drinking water reservoirs.

Stratified control of chemical crystallization in a pellet fluidized bed for pH-Adjusted fluoride and phosphate reduction: An experimental study.

Chemical crystallization granulation in a fluidized bed offers an environmentally friendly technology with significant promise for fluoride removal. This study investigates the impact of stratified pH control in a crystallization granulation fluidized bed for the removal of fluoride and phosphate on a pilot scale. The results indicate that using dolomite as a seed crystal, employing sodium dihydrogen phosphate (SDP) and calcium chloride as crystallizing agents, and controlling the molar ratio n(F):n(P):n(Ca) = 1:5:10 with an upflow velocity of 7.52 m/h, effectively removes fluoride and phosphate. Stratified pH control-maintaining weakly acidic conditions (pH = 6-7) at the bottom and weakly alkaline conditions (pH = 7-8) at the top-facilitates the induction of fluoroapatite (FAP) and calcium phosphate crystallization. This approach reduces groundwater fluoride levels from 9.5 mg/L to 0.2-0.6 mg/L and phosphate levels to 0.1-0.2 mg/L. Particle size analysis, scanning electron microscopy-energy-dispersive X-ray spectroscopy, and X-ray diffraction physical characterizations reveal significant differences in crystal morphology between the top and bottom layers, with the lower layer primarily generating high-purity FAP crystals. Further analysis shows that dolomite-induced FAP crystallization offers distinct advantages. SDP not only dissolves on the dolomite surface to provide active sites for crystallization but also, under weakly acidic conditions, renders both dolomite and FAP surfaces negatively charged. This allows for the effective adsorption of PO43-, HPO42-, and F- anions onto the crystal surfaces. This study provides supporting data for the removal of fluoride from groundwater through induced FAP crystallization in a chemical crystallization pellet fluidized bed.

Induced crystallization for the simultaneous removal of hardness-iron-manganese in groundwater: An experimental study.

Hardness, iron, and manganese are common groundwater pollutants, that frequently surpass the established discharge standard concentrations. They can be effectively removed, however, through induced crystallization. This study has investigated the effectiveness of the simultaneous removal of hardness-iron-manganese and the crystallization kinetics of calcium carbonate during co-crystallization using an automatic potentiometric titrator. The impacts pH, dissolved oxygen (DO), and ion concentration on the removal efficiency of iron and manganese and their influence on calcium carbonate induced crystallization were assessed. The results suggest that pH exerts the most significant influence during the removal of hardness, iron, and manganese, followed by DO, and then the concentration of iron and manganese ions. The rate of calcium carbonate crystallization increased with pH, stabilizing at a maximum of 10-10 m/s. Iron and manganese can be reduced from an initial level of 4 mg/L to <0.3 mg/L and 0.1 mg/L, respectively. The removal rate of iron, however, was notably higher than that of manganese. The DO concentration correlates positively with the removal of iron and manganese but has minimal impact on the calcium carbonate crystallization process. During the removal of iron and manganese, competitive interactions occur with the substrate, as increases in the concentration of one ion will inhibit the removal rate of the other. Characterization of post-reaction particles and mechanistic analysis reveals that calcium is removed through the crystallization of CaCO3, while most iron is removed through precipitation as Fe2O3 and FeOOH. Manganese is removed via two mechanisms, crystallization of manganese oxide (MnO2/Mn2O3) and precipitation. Overall, this research studies the removal efficiency of coexisting ions, the crystallization rate of calcium carbonate, and the mechanism of simultaneous removal, and provides valuable data to aid in the development of new removal techniques for coexisting ions.

Comparing Mn-based oxides filters started by KMnO4 versus K2FeO4 for ammonium and manganese removal: Formation mechanism of active species.

A pilot-scale filtration system was adopted to prepare filter media with catalytic activity to remove manganese (Mn2+) and ammonium (NH4+-N). Three different combinations of oxidants (KMnO4 and K2FeO4) and reductants (MnSO4 and FeCl2) were used during the start-up period. Filter R3 started up by KMnO4 and FeCl2 (Mn7+→MnOx) exhibited excellent catalytic property, and the NH4+-N and Mn2+ removal efficiency reached over 80% on the 10th and 35th days, respectively. Filter R1 started up by K2FeO4 and MnSO4 (MnOx←Mn2+) exhibited the worst catalytic property. Filter R2 started up by KMnO4 and MnSO4 (Mn7+→MnOx←Mn2+) were in between. According to Zeta potential results, the Mn-based oxides (MnOx) formed by Mn7+→MnOx performed the highest pHIEP and pHPZC. The higher the pHIEP and pHPZC, the more unfavorable the cation adsorption. However, it was inconsistent with its excellent Mn2+ and NH4+-N removal abilities, implying that catalytic oxidation played a key role. Combined with XRD and XPS analysis, the results showed that the MnOx produced by the reduction of KMnO4 showed early formation of buserite crystals, high degree of amorphous, high content of Mn3+ and lattice oxygen with the higher activity to form defects. The above results showed that MnOx produced by the reduction of KMnO4 was more conducive to the formation of active species for catalytic oxidation of NH4+-N and Mn2+ removal. This study provides new insights on the formation mechanisms of the active MnOx that could catalytic oxidation of NH4+-N and Mn2+.

Water-Stabilized Vanadyl Phosphate Monohydrate Ultrathin Nanosheets toward High Voltage Al-Ion Batteries.

Al ion batteries (AIBs) are attracting considerable attention owing to high volumetric capacity, low cost, and high safety. However, the strong electrostatic interaction between Al3+ and host lattice leads to discontented cycling life and inferior rate capability. Herein, a new strategy of employing water molecules contained VOPO4 ·H2 O to boost Al3+ migration via the charge shielding effect of water is reported. It is revealed that VOPO4 ·H2 O with water lubrication effect and smaller steric hindrance owns high capacity and fast Al3+ diffusion, while the loss of unstable water upon cycling leads to a rapid performance degradation. To address this problem, ultrathin VOPO4 ·H2 O@MXene nanosheets are fabricated via the formed TiOV bond between VOPO4 ·H2 O and MXene. The MXene aided exfoliation results in enhanced VOwater bond strength between H2 O and VOPO4 that endows the obtained composite with strong water holding ability, contributing to the extraordinary cycling stability. Consequently, the VOPO4 ·H2 O@MXene delivers a high discharge potential of 1.8 V and maintains discharge capacities of 410 and 374.8 mAh g-1 after 420 and 2000 cycles at the current densities of 0.5 and 1.0 A g-1 , respectively. This work provides a new understanding of water-contained AIBs cathodes and vital guidance for developing high-performance AIBs.

Hybrid MOF Template-Directed Construction of Hollow-Structured In2 O3 @ZrO2 Heterostructure for Enhancing Hydrogenation of CO2 to Methanol.

Direct hydrogenation of CO2  to methanol using green hydrogen has emerged as a promising method for carbon neutrality, but qualifying catalysts represent a grand challenge. In2 O3 /ZrO2  catalyst has been extensively applied in methanol synthesis due to its superior activity; however, the electronic effect by strong oxides-support interactions between In2 O3  and ZrO2  at the In2 O3 /ZrO2  interface is poorly understood. In this work, abundant In2 O3 /ZrO2  heterointerfaces are engineered in a hollow-structured In2 O3 @ZrO2  heterostructure through a facile pyrolysis of a hybrid metal-organic framework precursor MIL-68@UiO-66. Owing to well-defined In2 O3 /ZrO2  heterointerfaces, the resultant In2 O3 @ZrO2  exhibits superior activity and stability toward CO2  hydrogenation to methanol, which can afford a high methanol selectivity of 84.6% at a conversion of 10.4% at 290 °C, and 3.0 MPa with a methanol space-time yield of up to 0.29 gMeOH  gcat -1  h-1 . Extensive characterization demonstrates that there is a strong correlation between the strong electronic In2 O3 -ZrO2  interaction and catalytic selectivity. At In2 O3 /ZrO2  heterointerfaces, the electron tends to transfer from ZrO2  to In2 O3  surface, which facilitates H2  dissociation and the hydrogenation of formate (HCOO*) and methoxy (CH3 O*) species to methanol. This study provides an insight into the In2 O3 -based catalysts and offers appealing opportunities for developing heterostructured CO2  hydrogenation catalysts with excellent activity.

Weak-value amplified surface plasmon resonance sensor based on joint detection of optical activity and refractive index.

A versatile system combining surface plasmon resonance (SPR) and weak value amplification (WVA) is presented, which can measure the optical activity and refractive index of chiral/achiral molecules, ionic compounds, and their mixture in solution individually or simultaneously. The variations in output light intensity directly exhibit high sensitivity to changes in optical activity and refractive index of the aforementioned substances. Furthermore, by examining the correlation between the intensity variation trend and the optical activity of the chiral molecule, the molecule's absolute configuration can be ascertained. Utilizing this instrument, optical rotation with a resolution of 3.04 × 10-6 rad and refractive index with a resolution of 5.57 × 10-9 RIU were obtained. As an attempt at practical application, this sensor was used to detect the adulteration of glucose and fructose in pure honey. Not only can such compromised honey be distinguished from pure honey using the refractive index or optical rotation, but the difference in optical activity can also be employed to effectively differentiate between adulterated honey samples containing glucose and fructose separately.

Keyframe-aided resolution enhancement network for dynamic super-resolution structured illumination microscopy.

Deep learning has been used to reconstruct super-resolution structured illumination microscopy (SR-SIM) images with wide-field or fewer raw images, effectively reducing photobleaching and phototoxicity. However, the dependability of new structures or sample observation is still questioned using these methods. Here, we propose a dynamic SIM imaging strategy: the full raw images are recorded at the beginning to reconstruct the SR image as a keyframe, then only wide-field images are recorded. A deep-learning-based reconstruction algorithm, named KFA-RET, is developed to reconstruct the rest of the SR images for the whole dynamic process. With the structure at the keyframe as a reference and the temporal continuity of biological structures, KFA-RET greatly enhances the quality of reconstructed SR images while reducing photobleaching and phototoxicity. Moreover, KFA-RET has a strong transfer capability for observing new structures that were not included during network training.

Xanthones from securidaca inappendiculata antagonizes the anti-rheumatic effect of methotrexate by inhibiting reduced folate carrier 1.

BACKGROUND: The first-line anti-rheumatic drug methotrexate (MTX) is used in the combination. Because of the unpredictable adverse reactions, optimization of relevant regimens is necessary and meaningful. This study aimed to study the possible interaction between Securidaca inappendiculate Hassk. Derived xanthones and MTX. METHODS: We established adjuvant-induced arthritis (AIA) model, which was treated with MTX and MTX + xanthone-rich fraction (XRF). The clinical efficacy was evaluated by histopathological examination, and LC-MS was used to monitor the blood concentration of MTX. Western blotting and immunohistochemistry were used to detect protein expression. In vitro, we assessed the activity of related transporters by cellular uptake assay based on HEK-293T cells. RESULTS: Compared with MTX-treated rats, inflammation in the immunized rats in the MTX + XRF group was obvious, indicating that XRF antagonized the anti-rheumatic effect of MTX. Meanwhile, XRF reduced liver and kidney injuries caused by MTX in addition to MTX. Results from immunohistochemical and nappendiculat assays suggested that XRF may reduce uptake of MTX by down-regulating reduced folate carrier 1 (RFC1). CONCLUSION: This study indicated that XRF could reduce the plasma concentration of MTX by inhibiting the expression of RFC1, antagonize the therapeutic effect of MTX on AIA rats, and reduce its oral bioavailability. The combination of S. inappendiculate and MTX should be further optimized to achieve the goal of increasing efficiency and reducing toxicity.

Urban stormwater disinfection, quality variability during storage and influence on the freshwater algae: Implications for reuse safety.

Stormwater reuse is one of the most important ways to mitigate water resource shortage. However, urban stormwater contains many bacteria species, which threaten the reuse safety. Therefore, stormwater disinfection is highly needed. Although disinfection has been widely conducted in the drinking water and reclaimed water, it is rarely carried out for stormwater. This study collected the roof stormwater and undertook chlorination disinfection. Two typical bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S.aureus) were selected in this study to investigate the disinfection efficiency. It is found that bacteria species present in the stormwater had an important influence on disinfection efficiency while the original stormwater quality did not show an obvious affect. However, when the disinfected stormwater was stored, the stormwater quality was highly variable during its storage process and the variability was affected by bacteria species. The S.aureus containing stormwater showed a high variability of quality and S.aureus significantly regrew. However, the E.coli containing stormwater quality had a relatively low variability and E.coli did not significantly regrew. Additionally, it is noted that after storage, the dissolved form of stormwater was more positive to the freshwater algae's growth while the particulate form (including bacteria and other particulate matters) was less. This implies that a further treatment such as filtration is needed before the stored stormwater is recharged into receiving waters in order to remove particulate forms. These research outcomes can provide useful insight to effective stormwater disinfection and ensure reuse safety.

Improved Dye Survival in Expansion Microscopy through Stabilizer-Conjugated Linkers.

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer-containing organic fluorophores in ExM, as a mitigation strategy for this radical-induced dye degradation. Through direct conjugation of 4-nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore-conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer-modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.