Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging.

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution.

Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

Biological Insight from Super-Resolution Microscopy: What We Can Learn from Localization-Based Images.

Super-resolution optical imaging based on the switching and localization of individual fluorescent molecules [photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc.] has evolved remarkably over the last decade. Originally driven by pushing technological limits, it has become a tool of biological discovery. The initial demand for impressive pictures showing well-studied biological structures has been replaced by a need for quantitative, reliable data providing dependable evidence for specific unresolved biological hypotheses. In this review, we highlight applications that showcase this development, identify the features that led to their success, and discuss remaining challenges and difficulties. In this context, we consider the complex topic of defining resolution for this imaging modality and address some of the more common analytical methods used with this data.

Colorful low-emissivity paints for space heating and cooling energy savings.

Space heating and cooling consume ~13% of global energy every year. The development of advanced materials that promote energy savings in heating and cooling is gaining increasing attention. To thermally isolate the space of concern and minimize the heat exchange with the outside environment has been recognized as one effective solution. To this end, here, we develop a universal category of colorful low-emissivity paints to form bilayer coatings consisting of an infrared (IR)-reflective bottom layer and an IR-transparent top layer in colors. The colorful visual appearance ensures the aesthetical effect comparable to conventional paints. High mid-infrared reflectance (up to ~80%) is achieved, which is more than 10 times as conventional paints in the same colors, efficiently reducing both heat gain and loss from/to the outside environment. The high near-IR reflectance also benefits reducing solar heat gain in hot days. The advantageous features of these paints strike a balance between energy savings and penalties for heating and cooling throughout the year, providing a comprehensive year-round energy-saving solution adaptable to a wide variety of climatic zones. Taking a typical midrise apartment building as an example, the application of our colorful low-emissivity paints can realize positive heating, ventilation, and air conditioning energy saving, up to 27.24 MJ/m2/y (corresponding to the 7.4% saving ratio). Moreover, the versatility of the paint, along with its applicability to diverse surfaces of various shapes and materials, makes the paints extensively useful in a range of scenarios, including building envelopes, transportation, and storage.

Completing the canvas: advances and challenges for DNA-PAINT super-resolution imaging.

Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging.

Distinct SAP102 and PSD-95 Nano-organization Defines Multiple Types of Synaptic Scaffold Protein Domains at Single Synapses.

MAGUK scaffold proteins play a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. In particular, the MAGUKs SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform both overlapping and unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could underlie its unique functions and impact how SAP102 scaffolds synaptic proteins. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses in mixed-sex rat cultured neurons. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters (NCs). However, SAP102 NCs were smaller and denser than PSD-95 NCs across development. Additionally, only a subset of SAP102 NCs co-organized with PSD-95, revealing MAGUK nanodomains within individual synapses containing either one or both proteins. These MAGUK nanodomain types had distinct NC properties and were differentially enriched with the presynaptic release protein Munc13-1. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions.

Emerging accessibility patterns in long telomeric overhangs.

We present single-molecule experimental and computational modeling studies investigating the accessibility of human telomeric overhangs of physiologically relevant lengths. We studied 25 different overhangs that contain 4-28 repeats of GGGTTA (G-Tract) sequence and accommodate one to seven tandem G-quadruplex (GQ) structures. Using the FRET-PAINT method, we probed the distribution of accessible sites via a short imager strand, which is complementary to a G-Tract and transiently binds to available sites. We report accessibility patterns that periodically change with overhang length and interpret these patterns in terms of the underlying folding landscape and folding frustration. Overhangs that have [4n]G-Tracts, (12, 16, 20…) demonstrate the broadest accessibility patterns where the peptide nucleic acid probe accesses G-Tracts throughout the overhang. On the other hand, constructs with [4n+2]G-Tracts, (14, 18, 22…) have narrower patterns where the neighborhood of the junction between single- and double-stranded telomeres is most accessible. We interpret these results as the folding frustration being higher in [4n]G-Tract constructs compared to [4n+2]G-Tract constructs. We also developed a computational model that tests the consistency of different folding stabilities and cooperativities between neighboring GQs with the observed accessibility patterns. Our experimental and computational studies suggest the neighborhood of the junction between single- and double-stranded telomeres is least stable and most accessible, which is significant as this is a potential site where the connection between POT1/TPP1 (bound to single-stranded telomere) and other shelterin proteins (localized on double-stranded telomere) is established.

Single-Molecule Assessment of DNA Hybridization Kinetics on Dye-Loaded DNA Nanostructures.

DNA nanostructures offer a versatile platform for precise dye assembly, making them promising templates for creating photonic complexes with applications in photonics and bioimaging. However, despite these advancements, the effect of dye loading on the hybridization kinetics of single-stranded DNA protruding from DNA nanostructures remains unexplored. In this study, the DNA points accumulation for imaging in the nanoscale topography (DNA-PAINT) technique is employed to investigate the accessibility of functional binding sites on DNA-templated excitonic wires. The results indicate that positively charged dyes on DNA frameworks can accelerate the hybridization kinetics of protruded ssDNA through long-range electrostatic interactions. Furthermore, the impacts of various charged dyes and binding sites are explored on diverse DNA frameworks with varying cross-sizes. The research underscores the crucial role of electrostatic interactions in DNA hybridization kinetics within DNA-dye complexes, offering valuable insights for the functionalization and assembly of biomimetic photonic systems.

Spectral Phasor Applied to Spectrally-Resolved Single Molecule Localization Microscopy.

Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.

Toxicity of Aged Paint Particles to Soil Ecosystems: Insights from Caenorhabditis elegans.

Despite the extensive global consumption of architectural paint, the toxicological effects of aged exterior paint particles on terrestrial biota remain largely uncharacterized. Herein, we assessed the toxic effect of aged paint particles on soil environments using the nematode Caenorhabditis elegans (C. elegans) as a test organism. Various types of paint particles were generated by fragmentation and sequential sieving (500-1000, 250-500, 100-250, 50-100, 20-50 µm) of paint coatings collected from two old residential areas. The paint particles exerted different levels of toxicity, as indicated by a reduction in the number of C. elegans offspring, depending on their size, color, and layer structure. These physical characteristics were found to be closely associated with the chemical heterogeneity of additives present in the paint particles. Since the paint particle sizes were larger than what C. elegans typically consume, we attributed the toxicity to leachable additives present in the paint particles. To assess the toxicity of these leachable additives, we performed sequential washings of the paint particles with distilled water and ethanol. Ethanol washing of the paint particles significantly reduced the soil toxicity of the hydrophobic additives, indicating their potential environmental risk. Liquid chromatography-mass spectrometry analysis of the ethanol leachate revealed the presence of alkyl amines, which exhibited a high correlation with the toxicity of the paint particles. Further toxicity testing using an alkyl amine standard demonstrated that a paint particle concentration of 1.2% in soil could significantly reduce the number of C. elegans offspring. Our findings provide insights into the potential hazards posed by aged paint particles and their leachable additives in the terrestrial environment.

Lead Speciation, Bioaccessibility, and Sources for a Contaminated Subset of House Dust and Soils Collected from Similar United States Residences.

Residential lead (Pb) exposure is of critical concern to families globally as Pb promotes severe neurological effects in children, especially those less than 5 years old, and no blood lead level is deemed safe by the US Center for Disease Control. House dust and soils are commonly thought to be important sources of Pb exposure. Probing the relationship between house dust and soil Pb is critical to understanding residential exposure, as Pb bioavailability is highly influenced by Pb sources and/or species. We investigated paired house dust and soil collected from homes built before 1978 to determine Pb speciation, source, and bioaccessibility with the primary goal of assessing chemical factors driving Pb exposure in residential media. House dust was predominately found to contain (hydro)cerussite (i.e., Pb (hydroxy)carbonate) phases commonly used in Pb-based paint that, in-turn, promoted elevated bioaccessibility (>60%). Pb X-ray absorption spectroscopy, µ-XRF mapping, and Pb isotope ratio analysis for house dust and soils support house dust Pb as chemically unique compared to exterior soils, although paint Pb is expected to be a major source for both. Soil pedogenesis and increased protection from environmental conditions (e.g., weathering) in households is expected to greatly impact Pb phase differences between house dust and soils, subsequently dictating differences in Pb exposure.

Graphene nanosheet reinforcement of polyurethane nanocomposite for green and sustainable photoluminescence, superhydrophobic, and anticorrosive paint.

A simple and environmentally friendly method was developed for smart and efficient waterborne polyurethane (PUR) paint. Sugarcane bagasse was recycled into reduced graphene oxide nanosheets (rGONSs). Both lanthanide-doped aluminate nanoparticles (LAN; photoluminescent agent, 7-9 nm) and rGONSs (reinforcement agent) were integrated into a waterborne polyurethane to produce a novel photoluminescent, hydrophobic, and anticorrosive nanocomposite coating. Using ferrocene-based oxidation under masked circumstances, graphene oxide nanosheets were produced from sugarcane bagasse. The oxidized semicarbazide (SCB) nanostructures were integrated into polyurethane coatings as a drying, anticorrosion, and crosslinking agent. Polyurethane coatings with varying amounts of phosphor pigment were prepared and subsequently applied to mild steel. The produced paints (LAN/rGONSs@PUR) were tested for their hydrophobicity, hardness, and scratch resistance. Commission Internationale de l'éclairage (CIE) Laboratory parameters and photoluminescence analysis established the opacity and colourimetric properties of the nanocomposite coatings. When excited at 365 nm, the luminescent transparent paints emitted a strong greenish light at 517 nm. The anticorrosion characteristics of the coated steel were investigated. The phosphor-containing (11% w/w) polyurethane coatings displayed the most pronounced anticorrosion capability and long-persistent luminosity. The prepared waterborne polyurethane paints were very photostable and durable.

Bubble Sensors for Temperature Measurements through a Colorimetric Approach.

This paper introduces an innovative sensor utilizing bubbles coated with thermochromic paint, aiming to facilitate temperature measurements in challenging-to-reach locations without the requirement of an external power source. The research conducted is innovative in terms of both methodology and application. The characterization of the thermochromic properties of paints was, in fact, performed using spectroradiometric measurements by selecting a temperature range useful for applications in various fields including preventive conservation. The study encompasses two main objectives: (1) analyzing the color characteristics of thermochromic paint and plastic resin that forms the bubbles, and (2) assessing a temperature sensor comprising a thermochromic paint-coated bubble subjected to temperature variations. The thermochromic paint exhibits reversible color modifications in response to temperature changes, making it an ideal candidate for applications of this nature. The color characterization phase involves measurements using a spectroradiometer to compare the spectral reflectance factor (SRF%) of the colored plastic resin spread on canvas with that of the inflated bubbles. The sensor characterization entails evaluating color changes of the thermochromic paint on the bubble surface with varying temperatures. Experimental results indicate that the combination of a red (R) bubble and blue (B) thermochromic paint produces quantifiable color variations suitable for the proposed applications, whereas the alternative combination under examination, namely a blue bubble and red thermochromic paint, yields less accurate results. Considering that for both thermochromic paints the color change temperature is 35 °C, it is possible to see how, for B bubble with R thermochromic paint, the chromatic coordinates change value: C* = 3.14 ± 0.14 and h = 289.54 ± 11.58 at room temperature, while C* = 2.96 ± 0.12 and h = 304.20 ± 12.17 at 35 °C. The same is true for R bubble with B thermochromic paint where C* = 25.31 ± 1.01 and h* = 285.05 ± 11.40 at room temperature, while C* = 20.87 ± 0.85 and h = 288.37 ± 11.53 at 35 °C. The study demonstrates the potential of the approach and suggests further investigations into reproducibility and expanded color combinations. The results provide a promising basis for future improvements in temperature monitoring with thermochromic bubble sensors.

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.

Effect of Copper Antifouling Paint on Marine Degradation of Polypropylene: Uneven Distribution of Microdebris between Nagasaki Port and Goto Island, Japan.

Microplastics (MP) encompass not only plastic products but also paint particles. Marine microdebris, including MP, was retrieved from five sampling stations spanning Nagasaki-Goto island and was classified into six types, primarily consisting of MP (A), Si-based (B), and Cu-based (C) paint particles. Type-A particles, i.e., MP, were exceedingly small, with 74% of them having a long diameter of 25 µm or less. The vertical distribution of type C, containing cuprous oxide, exhibited no depth dependence, with its dominant size being less than 7 µm. It was considered that the presence of type C was associated with a natural phenomenon of MP loss. To clarify this, polypropylene (PP) samples containing cuprous oxide were prepared, and their accelerated degradation behavior was studied using a novel enhanced degradation method employing a sulfate ion radical as an initiator. Infrared spectroscopy revealed the formation of a copper soap compound in seawater. Scanning electron microscopy/energy-dispersive X-ray spectroscopy analysis indicated that the chemical reactions between Cl- and cuprous oxide produced Cu+ ions. The acceleration of degradation induced by the copper soap formed was studied through the changes in the number of PP chain scissions, revealing that the presence of type-C accelerated MP degradation.

Super-Resolved Protein Imaging Using Bifunctional Light-Up Aptamers.

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila.

Single-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. Here, we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies.

Insights into Health Risks of Face Paint Application to Opera Performers: The Release of Heavy Metals and Stage-Light-Induced Production of Reactive Oxygen Species.

Face paints used by opera performers have been shown to contain high levels of heavy metals. However, whether frequent exposure, via dermal contact and inadvertent oral ingestion, results in occupational diseases is unknown, as is the potential exacerbation of toxicity by high-intensity irradiation from stage lights. In this study, we examined the release of Cr, Cu, Pb, and Zn from 40 face paints and the consequent health risks posed by different practical scenarios involving their use. The results showed that the in vitro bioaccessibility (IVBA) of Cr, Cu, Pb, and Zn in the tested products was, on average, 7.0, 5.5, 19.9, and 7.9% through oral ingestion and 1.1, 2.2, 1.6, and 1.2% through dermal contact, respectively. Stage light irradiation significantly increased the IVBA associated with dermal contact, to the average of 4.8, 34.9, 5.7, and 1.9% for Cr, Cu, Pb, and Zn, respectively. The increase was mainly due to the light-induced generation of reactive oxygen species, particularly hydroxyl free radicals. The vitality and transcriptional response of 3D skin models as well as a quantitative risk assessment of skin sensitization indicated that dermal contact with face paints may induce predictable skin damage and potentially other skin diseases. Long-term exposure to face paints on stage may also pose a non-carcinogenic health risk. The demonstrated health risks to opera performers of face paint exposure should lead to strict regulations regarding the content of theatrical face paints.