Publications

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

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

© 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

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