Conventional fluorophores display strongly environment-dependent fluorescence properties, which limits their performance under the oxidizing, high-density and complex interfacial conditions characteristic of advanced imaging experiments. We show that rylene dyes can function as environment-tolerant blinking fluorophores whose intermittency arises from reversible photoinduced charge transfer between the chromophore and ubiquitous environmental acceptors such as oxygen, rather than from specialized buffer chemistries. We then demonstrate that the apparent fluorescence loss under OsO 4 staining arises mainly from static quenching and inner‑filter effects, not from irreversible chemical degradation, and we account for this by a unified model combining distance‑dependent photoinduced electron transfer/FRET with absorption by reduced osmium species. This mechanistic picture explains why rylene emitters remain functional after standard OsO 4 staining and epoxy resin embedding, and yields design rules for fluorophores that retain blinking in such extreme environments. Leveraging this understanding, we label these dyes to biomolecular targets and demonstrate the first dual-color super‑resolution correlative light and electron microscopy on the same section. By integrating mechanistic insight with performance in a stringent application, this work establishes a rational framework for designing next-generation probes for advanced correlative nanoscopy.

We present a registration strategy that targets cross-modality images of different resolutions. Multimodal registration with reference to the prediction of landmarks is still a very challenging problem. Many model-based predictions and manual identification of landmarks have been studied and explored with the use of supervised methods. Despite the various methods available, they all necessitate some level of human involvement, hindering the swift and automated registration process. Here, we demonstrate the image registration of correlative light and electron microscopy (CLEM) images with the precise localization of landmark positions using a training-free, unsupervised neural network algorithm, which can process image sizes up to 11k pixels x 11k pixels, depending on the GPU memory size.

The complex dynamics of synthetic supramolecular systems in living cellular environments impede the correlation between the transient hierarchical species and their biological functions. Achieving this correlation demands a breakthrough that combines the precise control of supramolecular events at discrete time points via synthetic chemistry with their real-time visualization in native cells. In the present study, we reported two peptide sequences that undergo visible light-induced molecular and supramolecular transformations to form various assembly species in cells. In contrast to endogenous stimulus-responsive assembly, the proposed photochemistry enables full control over the photolysis reaction where the monomer generation and local concentration regulate the subsequent assembly kinetics. Phasor-fluorescence lifetime imaging traced the formation of various assembly states in cells associated with monomer activation and consumption, whereas correlative light-electron microscopy revealed the intracellular nanofibres formed. The temporally resolved assembly process shows that the emergence of cytotoxicity correlates with the accumulation of oligomers beyond the cellular efflux threshold. Spatiotemporal control over the assembly of intracellular peptide nanofibres using visible light is reported. The photolysis-promoted monomer generation correlates the structure formation kinetics to the toxicity in cells.

In the field of nano-bio interactions, the cellular uptake of nanocarriers and the modulating factors, e.g. the presence of a protein shell - the protein corona - adsorbed on the nanocarrier surface are well studied. However, little is known about the detailed mechanisms by which nanocarriers leave a cell - the exocytosis processes of nanocarriers from cells - and their driving determinants. Here, we analyzed the protein components of magnetic dextran-coated iron oxide particles exocytosed from a human tumor cell model over time using mass spectrometry-based proteomics and bioinformatic annotation analysis to map the dynamics of the nanocarrier exocytosis mechanism. We demonstrated that the protein corona desorbed from exocytosed particles can serve as a molecular fingerprint of the exocytosis pathways involved. We correlated the proteomic findings mainly using ultrastructural imaging, but also with other conventional methods such as inductively coupled plasma optical emission spectrometry, flow cytometry, and confocal microscopy. Our data showed that the presence of a pre-coated human plasma protein corona did not drastically affect the extent and route of exocytosis. Most importantly, we demonstrated the time-dependency of a non-conventional exocytosis mechanism for the iron oxide particles investigated with a major contribution of lysosomal exocytosis accompanied by secretion of extracellular vesicles and extracellular vesicle proteins. STATEMENT OF SIGNIFICANCE: Nanocarriers are not only internalized by cells but also undergo exocytosis. We show that their intracellular fate can be traced via protein signatures on their surface post-exocytosis. Analyzing this protein corona is challenging due to limited nanoparticle recovery, but label-free quantitative liquid chromatography-mass spectrometry (LC-MS) enabled identification of pathway-specific protein fingerprints. Clustering these fingerprints revealed distinct intracellular trafficking routes and exocytosis mechanisms, highlighting the complexity of both processes. Transmission electron microscopy (TEM) validated nanocarrier transit through cellular compartments. Notably, we observed time-dependent involvement of an unconventional exocytosis mechanism, predominantly lysosomal-like exocytosis. This integrative approach deciphers nanocarrier behavior, linking surface biomolecular profiles to intracellular dynamics.

Characterising the size and morphology of nanoparticles (NPs), especially in complex systems like core-shell particles and nanocapsules, remains a significant challenge due to limitations in resolution and applicability of traditional methods. Here, we explore a novel approach to image-based NP characterisation using 2D class averaging (2D-CA) techniques used in single particle analysis. By leveraging well-established software originally developed in structural biology, our method provides detailed size distribution analysis for diverse NP systems, including bimodal particle size distributions, nanocapsules and nanorods. To validate the efficacy and accuracy of this technique, we conduct a comparative study against established characterisation methods, highlighting the potential of 2D-CA to enhance the analysis of challenging NP systems that are otherwise inaccessible using conventional methods, such as highly agglomerated NPs. Our results indicate that single particle averaging techniques offer a sound statistical basis for NP size distribution determination, coupled with a streamlined workflow that utilises established software. This method facilitates the processing of large numbers of micrographs, yielding statistically robust results with minimal human bias through automated particle identification.

Supramolecular assemblies found in nature demonstrate the concept of creating functionality through structure formation. In recent years, these complex natural architectures have inspired the development of materials for the formation of synthetic nanostructures within living cells. These intracellular assemblies have the potential to modulate cellular processes, yet their specific effects on cellular metabolism and 3D cell networks, such as tumor spheroids, still remain underexplored. Herein, the study correlates the glutathione-induced formation of synthetic nanostructures inside MDA-MB-231 triple-negative breast cancer cells to the metabolic disruption and mitochondrial degradation observed in 2D cell culture, as well as to cell death and size decrease in a 3D tumor spheroid model. In 2D cell culture, material-cell interactions are examined through live-cell imaging and by quantifying changes in mitochondrial respiration. By studying the interplay between glutathione-responsive cytosolic peptide assembly and the implications on the integrity of the mitochondrial network, as well as on 3D cell networks, the work advances the understanding of how synthetic intracellular nanofibers impact vital functions of living cells.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Macromolecular crowding agents, such as poly(ethylene glycol) (PEG), are often used to mimic cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, we demonstrate and quantitatively explore the diverse effects of PEG on the phase separation and maturation of protein condensates. We use two model proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with a well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying interactions, which hasten beta-sheet formation and subsequent droplet gelation. In contrast to BSA, PEG enhances droplet stability and limits the available solvent for protein solubilization, inducing only minimal changes in the secondary structure, pointing toward a significantly different role of the crowding agent. Strikingly, we detect almost no presence of PEG in Nup droplets, whereas PEG is moderately detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins; PEG can accelerate protein maturation in liquid-liquid phase separation systems, but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.

Journal Article Multi-resolution Cross-modality Image Registration Using Unsupervised Deep Learning Approach Get access Daksh Daksh, Daksh Daksh Max Planck Institute for Polymer Research, Mainz, Germany Corresponding author: daksh@mpip-mainz.mpg.de Search for other works by this author on: Oxford Academic Google Scholar Anke Kaltbeitzel, Anke Kaltbeitzel Max Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Katharina Landfester, Katharina Landfester Max Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Ingo Lieberwirth Ingo Lieberwirth Max Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1964–1965, https://doi.org/10.1093/micmic/ozad067.1017 Published: 22 July 2023

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.

Typically, 2D nanosheets have a homogeneous surface, making them a major challenge to structure. This study proposes a novel concept of 2D organic nanosheets with a heterogeneously functionalized surface. This work achieves this by consecutively crystallizing two precisely synthesized polymers with different functional groups in the polymer backbone in a two-step process. First, the core platelet is formed and then the second polymer is crystallized around it. As a result, the central area of the platelets has a different surface functionality than the periphery. This concept offers two advantages: the resulting polymeric 2D platelets are stable in dispersion, which simplifies further processing and makes both crystal surfaces accessible for subsequent functionalization. Additionally, a wide variety of polymers can be used, making the process and the choice of surface functionalization very flexible.

Journal Article Correlative Microscopy for the Identification of Intracellular Nanoparticles and their Cellular Processing Get access Ingo Lieberwirth, Ingo Lieberwirth Max-Planck Institute for Polymer Research, Mainz, Germany Corresponding author: lieberw@mpip-mainz.mpg.de Search for other works by this author on: Oxford Academic Google Scholar Shen Han, Shen Han Max-Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Anke Kaltbeitzel, Anke Kaltbeitzel Max-Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Gunnar Glaßer, Gunnar Glaßer Max-Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Katharina Landfester Katharina Landfester Max-Planck Institute for Polymer Research, Mainz, Germany Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Page 1955, https://doi.org/10.1093/micmic/ozad067.1012 Published: 22 July 2023

The complex dynamics and transience of molecular aggregation pathways leading to amyloid-based neurodegeneration complicate the mechanistic understanding of these fatal diseases. Current technologies are unable to track the molecular processes leading to the onset of amyloidogenesis where real-time information is imperative to correlate its rich biology. Using a chemically-designed amyloidogenic molecule, we map its molecular transformation into amyloids and the resultant fusion with endosomes to form discrete, hollow plaque clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy/tomography (CLEM), spatiotemporal splicing of the aggregation events shows time-correlated respiratory failure. We reveal that the initial dynamics of aggregation invokes cellular responses on a systemic level.

Nanostructure-based functions are omnipresent in nature and essential for the diversity of life. Unlike small molecules, which are often inhibitors of enzymes or biomimetics with established methods of elucidation, we show that functions of nanoscale structures in cells are complex and can implicate system-level effects such as the regulation of energy and redox homeostasis. Herein, we design a platinum(II)-containing tripeptide that assembles into intracellular fibrillar nanostructures upon molecular rearrangement in the presence of endogenous H2O2. The formed nanostructures blocked metabolic functions, including aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. As a consequence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated. We demonstrate that assembly-driven nanomaterials offer a rich avenue to achieve broad-spectrum bioactivities that could provide new opportunities in drug discovery.

This work analyzes the intracellular fate of protein-based nanocarriers along their endolysosomal pathway by means of correlative light and electron microscopy methods. To unambiguously identify the nanocarriers and their degradation remnants in the cellular environment, they are labeled with fluorescent, inorganic nanoplatelets. This allows tracking the nanocarriers on their intracellular pathway by means of electron microscopy imaging. From the present data, it is possible to identify different cell compartments in which the nanocarriers are processed. Finally, three different terminal routes for the intracellular destiny of the nanocarriers are presented. These findings are important to reveal the degradation process of protein nanocapsules and contribute to the understanding of the therapeutic success of an encapsulated drug.

Nanostructure-based functions are omnipresent in biology and essential for the diversity of life. Despite their importance, it is difficult to establish mechanisms that define their bioactivity and rationalize them through synthetic designs. As such, strategies that connect bioactive functions through structure formation are scarce. Herein, we design a near-infrared emitting platinum (II)-tripeptide that undergoes a rearrangement using endogenous H2O2 to rapidly assemble into fibrillar superstructures. The resultant assembly inhibits the metabolism of aggressive metastatic MDA-MB-231 cells and A549 cells at the systemic level by blocking aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. Hence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated, leading to apoptosis. By demonstrating that assembly-driven functions can inhibit broad biological pathways, supramolecular nanostructures could offer the next generation biomedical solutions beyond conventional applications.

Wet and dry foams are prevalent in many industries, ranging from the food processing and commercial cosmetic sectors to industries such as chemical and oil-refining. Uncontrolled foaming results in product losses, equipment downtime or damage and cleanup costs. To speed up defoaming or enable anti-foaming, liquid oil or hydrophobic particles are usually added. However, such additives may need to be later separated and removed for environmental reasons and product quality. Here, we show that passive defoaming or active anti-foaming is possible simply by the interaction of foam with chemically or morphologically modified surfaces, of which the superamphiphobic variant exhibits superior performance. They significantly improve retraction of highly stable wet foams and prevention of growing dry foams, as quantified for beer and aqueous soap solution as model systems. Microscopic imaging reveals that amphiphobic nano-protrusions directly destabilize contacting foam bubbles, which can favorably vent through air gaps warranted by a Cassie wetting state. This mode of interfacial destabilization offers untapped potential for developing efficient, low-power and sustainable foam and froth management.

In this work, we unravel the role of surface properties of colloidal particles on the formation of supraparticles (clusters of colloidal particles) in a colloidal Ouzo droplet. Self-lubricating colloidal Ouzo droplets are an efficient and simple approach to form supraparticles, overcoming the challenge of the coffee stain effect in situ. Supraparticles are an efficient route to high-performance materials in various fields, from catalysis to carriers for therapeutics. Yet, the role of the surface of colloidal particles in the formation of supraparticles using Ouzo droplets remains unknown. Therefore, we used silica particles as a model system and compared sterically stabilized versus electrostatically stabilized silica particles-positively and negatively charged. Additionally, we studied the effect of hydration. Hydrated negatively charged silica particles and sterically stabilized silica particles form supraparticles. Conversely, dehydrated negatively charged silica particles and positively charged amine-coated particles form flat film-like deposits. Notably, the assembly process is different for all the four types of particles. The surface modifications alter (a) the contact line motion of the Ouzo droplet and (b) the particle-oil and particle-substrate interactions. These alterations modify the particle accumulation at the various interfaces, which ultimately determines the shape of the final deposit. Thus, by modulating the surface properties of the colloidal particles, we can tune the shape of the final deposit, from a spheroidal supraparticle to a flat deposit. In the future, this approach can be used to tailor the supraparticles for applications such as optics and catalysis, where the shape affects the functionality.

To understand the removal of particles from surfaces by water drops, we used an inverted laser scanning confocal microscope to image the collision between a water drop and a particle on a flat polydimethylsiloxane (PDMS) surface. The dynamic drop-particle contact line was monitored by fixing the drop directly above the objective lens while moving the sample stage at well-defined speeds (10-500 μm s-1). The lateral force acting on the drop during the collision was measured as a function of speed, using a force sensor mounted on the microscope. Depending on the collision speed, the particle either stays attached at the rear of the drop or detaches from it. We propose a criterion to determine whether the particle remains attached to the drop based on the capillary and resistive forces acting on the particle during the collision. The forces measured when the particle crosses the air-water interface are compared to existing models. We adapted these to account for rolling of the particle. By comparing our experimental measurements with an analytical model for the capillary torque acting on a particle rolling at an interface, we provide detailed insights on the origins of the resistive force acting on the particle when it is pushed or pulled by the drop. A low friction force between the surface and the particle increases the likelihood of particle removal.