Publications

Supramolecular systems chemistry has been an area of active research to develop nanomaterials with life-like functions. Progress in systems chemistry relies on our ability to probe the nanostructure formation in solution. Often visualizing the dynamics of nanostructures which transform over time is a formidable challenge. This necessitates a paradigm shift from dry sample imaging towards solution-based techniques. We review the application of state-of-the-art techniques for real-time, in situ visualization of dynamic self-assembly processes. We present how solution-based techniques namely optical super-resolution microscopy, solution-state atomic force microscopy, liquid-phase transmission electron microscopy, molecular dynamics simulations and other emerging techniques are revolutionizing our understanding of active and adaptive nanomaterials with life-like functions. This Review provides the visualization toolbox and futuristic vision to tap the potential of dynamic nanomaterials.© 2022 Wiley-VCH GmbH.

JTD Keywords: electron-microscopy, fluorescence microscopy, in-situ, mechanical-properties, molecular simulations, nanostructures, polymerization, polymers, stimulated-emission, super-resolution microscopy, supramolecular chemistry, systems chemistry, water, Atomic-force microscopy, Liquid tem, Nanostructures, Super-resolution microscopy, Supramolecular chemistry, Systems chemistry

Characterization of the number and distribution of biological molecules on 2D surfaces is of foremost importance in biology and biomedicine. Synthetic surfaces bearing recognition motifs are a cornerstone of biosensors, while receptors on the cell surface are critical/vital targets for the treatment of diseases. However, the techniques used to quantify their abundance are qualitative or semi-quantitative and usually lack sensitivity, accuracy, or precision. Detailed herein a simple and versatile workflow based on super-resolution microscopy (DNA-PAINT) was standardized to improve the quantification of the density and distribution of molecules on synthetic substrates and cell membranes. A detailed analysis of accuracy and precision of receptor quantification is presented, based on simulated and experimental data. We demonstrate enhanced accuracy and sensitivity by filtering out non-specific interactions and artifacts. While optimizing the workflow to provide faithful counting over a broad range of receptor densities. We validated the workflow by specifically quantifying the density of docking strands on a synthetic sensor surface and the densities of PD1 and EGF receptors (EGFR) on two cellular models.

JTD Keywords: binding, biosensors, cancer, expression, kinetics, localization microscopy, quantification, receptors, single-molecule, super-resolution microscopy, Biosensors, Dna-paint, Quantification, Receptors, Single-molecule, Super-resolution microscopy, Superresolution microscopy

Antibody-functionalized nanoparticles (NPs) have shown numerous benefits in drug delivery and biosensing, improving the specificity of cell targeting and analyte detection, respectively. However, one of the main challenges is the lack of control over antibody orientation on the NP surface. Popular and easy conjugation strategies, such as carbodiimide-based conjugations, lead to a random orientation of antibodies on the NPs, compromising ligand functionality and contributing to undesired biological effects and reduced target recognition. While new methods for more controlled NP functionalization have been proposed, there is a lack of techniques that can elucidate the orientation of the antibodies at the single-particle level to determine the conjugation outcome and, therefore, the NPs' potential in selective targeting. Here, spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM), an optical super-resolution technique, is introduced to quantify the relationship between total and functional NP conjugated cetuximab antibodies at the single-particle level. An evident single-particle heterogeneity in total and functional cetuximab is observed, leading to particles with different functional : total ratios. Additionally, the results indicate that the functional : total ratio of cetuximab highly depends on the conjugated cetuximab concentration. Overall, SR-dSTORM represents a direct approach for the NP structure-functionality relationship quantification, providing a platform to improve antibody-conjugated NPs characterization and facilitating their rational design.

JTD Keywords: Heterogeneity, Superresolution microscopy

Enzyme-powered micro- and nanomotors make use of biocatalysis to self-propel in aqueous media and hold immense promise for active and targeted drug delivery. Most (if not all) of these micro- and nanomotors described to date are fabricated using a commercially available enzyme, despite claims that some commercial preparations may not have a sufficiently high degree of purity for downstream applications. In this study, the purity of a commercial urease, an enzyme frequently used to power the motion of micro- and nanomotors, was evaluated and found to be impure. After separating the hexameric urease from the protein impurities by size-exclusion chromatography, the hexameric urease was subsequently characterized and used to functionalize hollow silica microcapsules. Micromotors loaded with purified urease were found to be 2.5 times more motile than the same micromotors loaded with unpurified urease, reaching average speeds of 5.5 ?m/s. After comparing a number of parameters, such as enzyme distribution, protein loading, and motor reusability, between micromotors functionalized with purified vs unpurified urease, it was concluded that protein purification was essential for optimal performance of the enzyme-powered micromotor.

JTD Keywords: canavalin, catalysis, delivery, dls, enhanced diffusion, enzyme, lipase immobilization, micromotors, self-propulsion, super-resolution microscopy, urease, Mesoporous silica nanoparticles, Micromotors, Super-resolution microscopy

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used routinely by laboratories across the globe. With these methods we can understand the structure, dynamics and function of novel molecular architectures and their relations with the desired performance, guiding the development of the next generation of materials. Moreover, one of the challenges in materials chemistry is the lack of reproducibility due to improper publishing of the sample preparation protocol. In this context, the recent adoption of the reporting standard MIRIBEL (Minimum Information Reporting in Bio–Nano Experimental Literature) for material characterization and details of experimental protocols aims to provide complete, reproducible and reliable sample preparation for the scientific community. Thus, MIRIBEL should be immediately adopted in publications by scientific journals to overcome this challenge. Besides current standard spectroscopy and microscopy techniques, there is a constant development of novel technologies that aim to help chemists unveil the structure of complex materials. Among them super-resolution microscopy (SRM), an optical technique that bypasses the diffraction limit of light, has facilitated the study of synthetic materials with multicolor ability and minimal invasiveness at nanometric resolution. Although still in its infancy, the potential of SRM to unveil the structure, dynamics and function of complex synthetic architectures has been highlighted in pioneering reports during the last few years. Currently, SRM is a sophisticated technique with many challenges in sample preparation, data analysis, environmental control and automation, and moreover the instrumentation is still expensive. Therefore, SRM is currently limited to expert users and is not implemented in characterization routines. This perspective discusses the potential of SRM to transition from a niche technique to a standard routine method for material characterization. We propose a roadmap for the necessary developments required for this purpose based on a collaborative effort from scientists and engineers across disciplines.

JTD Keywords: blinking, fluorophore, intramolecular spirocyclization, localization, nanoparticles, resolution limit, reveals, single-molecule fluorescence, stimulated-emission, Characterization techniques, Diffraction, Distributed computer systems, Environmental management, Information reporting, Material chemistry, Materials characterization, Minimum information, Optical reconstruction microscopy, Optical resolving power, Sample preparation, Structure dynamics, Structure functions, Super-resolution microscopy, Synthesized materials

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFluS). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

JTD Keywords: dna-paint, hemagglutinin, influenza, neuraminidase, paint, recombinant proteins, single-molecule localization microscopy, single-particle analysis, virus-like particles, Dna-paint, Hemagglutinin, Influenza, Neuraminidase, Paint, Recombinant proteins, Single particle analysis, Single-molecule localization microscopy, Single-particle analysis, Super-resolution microscopy, Superresolution microscopy, Virus-like particles

The importance of PEG architecture on nanoparticle (NP) functionality is known but still difficult to investigate, especially at a single particle level. Here, we apply DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), a super-resolution microscopy (SRM) technique, to study the surface functionality in poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs with different PEG structures. We demonstrated how the length of the PEG spacer can influence the accessibility of surface chemical functionality, highlighting the importance of SRM techniques to support the rational design of functionalized NPs.

JTD Keywords: chain-length, density, plga, surface, systems, Chain-length, Density, Dna, Microscopy technique, Nanoparticles, Nanoscale topography, Paint, Peg spacers, Plga, Poly lactide-co-glycolide, Poly-lactide-co-glycolide, Polyethylene glycols, Polylactide-co-glycolide, Single-particle, Super-resolution microscopy, Superresolution microscopy, Surface, Surface chemicals, Surface functionalities, Systems

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

JTD Keywords: cellular uptake, correlative light and electron microscopy (clem), density, electron microscopy (em), functionalization, heterogeneity, nanomedicine, nanoparticles, pegylation, plga, progress, quantification, size, Correlative light and electron microscopy (clem), Electron microscopy (em), Heterogeneity, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)

The characterization of polymeric materials is key towards the understanding of structure–activity relations and therefore for the rational design of novel and improved materials for a myriad of applications. Many microscopy techniques are currently used, with electron microscopy, fluorescence microscopy, and atomic force microscopy being the most relevant. In this perspective paper, we discuss the use of correlative imaging, that is, the combination of multiple imaging methodologies on the same sample, in the field of polymeric materials. This innovative approach is emerging as a powerful tool to unveil the structure and functional properties of biological and synthetic structures. Here we discuss the possibilities of correlative imaging and highlight their potential to answer open questions in polymer science.

JTD Keywords: correlative imaging, electron microscopy, material characterization, resolution microscopy, super‐, Atomic force microscopy, Correlative imaging, Electron microscopy, Material characterization, Super-resolution microscopy

© 2020 Elsevier Inc. Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

JTD Keywords: cells, click-chemistry, complex, correlative light and electron microscopy, cycloaddition, ligation, localization, proteins, resolution limit, single molecule localization microscopy, stochastic optical reconstruction microscopy (storm), storm, super-resolution microscopy, tokuyasu cryo-sectioning, tool, Click-chemistry, Correlative light and electron microscopy, Fluorescent-probes, Single molecule localization microscopy, Stochastic optical reconstruction microscopy (storm), Super-resolution microscopy, Tokuyasu cryo-sectioning, Transmission electron microscopy

© 2020 The Authors Although the concept of selective delivery has been postulated over 100 years ago, no targeted nanomedicine has been clinically approved so far. Nanoparticles modified with targeting ligands to promote the selective delivery of therapeutics towards a specific cell population have been extensively reported. However, the rational design of selective particles is still challenging. One of the main reasons for this is the lack of quantitative theoretical and experimental understanding of the interactions involved in cell targeting. In this review, we discuss new theoretical models and experimental methods that provide a quantitative view of targeting. We show the new advancements in multivalency theory enabling the rational design of super-selective nanoparticles. Furthermore, we present the innovative approaches to obtain key targeting parameters at the single-cell and single molecule level and their role in the design of targeting nanoparticles. We believe that the combination of new theoretical multivalent design and experimental methods to quantify receptors and ligands aids in the rational design and clinical translation of targeted nanomedicines.

JTD Keywords: binding-kinetics, biological identity, biomolecular corona, blood-brain-barrier, drug-delivery, gold nanoparticles, multivalency, nanotechnology, protein corona, quantitative characterization, rational design, super-selectivity, superresolution microscopy, tumor heterogeneity, Ligand-receptor interactions, Multivalency, Nanotechnology, Quantitative characterization, Rational design, Super-selectivity

The visualization of microtubules by combining optical and electron microscopy techniques provides valuable information to understand correlated intracellular activities. However, the lack of appropriate probes to bridge both microscopic resolutions restricts the areas and structures that can be comprehended within such highly assembled structures. Here, a versatile cyclometalated iridium (III) complex is designed that achieves synchronous fluorescence–electron microscopy correlation. The selective insertion of the probe into a microtubule triggers remarkable fluorescence enhancement and promising electron contrast. The long-life, highly photostable probe allows live-cell super-resolution imaging of tubulin localization and motion with a resolution of ≈30 nm. Furthermore, correlative light–electron microscopy and energy-filtered transmission electron microscopy reveal the well-associated optical and electron signal at a high specificity, with an interspace of ≈41 Å of microtubule monomer in cells.

JTD Keywords: Correlation light–electron microscopy, Microtubules, Organometallic probes, Super-resolution microscopy

Surface functionalization with targeting ligands confers to nanomaterials the ability of selectively recognize a biological target. Therefore, a quantitative characterization of surface functional molecules is critical for the rational development of nanomaterials-based applications, especially in nanomedicine research. Single-molecule localization microscopy can provide visualization of surface molecules at the level of individual particles, preserving the integrity of the material and overcoming the limitations of analytical methods based on ensemble averaging. Here we provide single-molecule localization data obtained on streptavidin-coated polystyrene particles, which can be exploited as a model system for surface-functionalized materials. After loading of the active sites of streptavidin molecules with a biotin-conjugated probe, they were imaged with a DNA-PAINT imaging approach, which can provide single-molecule imaging at subdiffraction resolution and molecule counting. Both raw records and analysed data, consisting in a list of space-time single-molecule coordinates, are shared. Additionally, Matlab functions are provided that analyse the single-molecule coordinates in order to quantify features of individual particles. These data might constitute a valuable reference for applications of similar quantitative imaging methodologies to other types of functionalized nanomaterials.

JTD Keywords: DNA-PAINT, Functional materials, Nanoparticles, Single-molecule localization microscopy, Super-resolution microscopy

Since their invention about two decades ago, super-resolution microscopes have become a method of choice in cell biology. Owing to a spatial resolution below 50 nm, smaller than the size of most organelles, and an order of magnitude better than the diffraction limit of conventional light microscopes, superresolution microscopy is a powerful technique for resolving intracellular trafficking. In this review we discuss discoveries in endocytosis and phagocytosis that have been made possible by super-resolution microscopy – from uptake at the plasma membrane, endocytic coat formation, and cytoskeletal rearrangements to endosomal maturation. The detailed visualization of the diverse molecular assemblies that mediate endocytic uptake will provide a better understanding of how cells ingest extracellular material.

JTD Keywords: Endocytosis, Endosomes, Organelles, Super-resolution microscopy, Trafficking

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

JTD Keywords: dSTORM, Gene delivery, Polyplexes, Stability, Super-resolution microscopy

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

JTD Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy