Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy.

Extracellular vesicles (EVs) play a key role in cell-cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm.

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology.

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples.

Bio-inspired microneedle design for efficient drug/vaccine coating.

Biomimetics is the interdisciplinary scientific field focused on the study and imitation of biological systems, with the aim of solving complex technological problems. In this paper, we present a new bio-inspired design for microneedles (MNs) and MN arrays, intended for rapidly coating the MNs with drug/vaccine. The biomimetic approach consists in ornamenting the lateral sides of pyramidal MNs with structures inspired by the external scent efferent systems of some European true bugs, which facilitate a directional liquid transport.

Biofunctionalization of Sub-Diffractionally Patterned Polymer Structures by Photobleaching.

Stimulated emission depletion (STED) nanolithography allows nanofabrication below the diffraction limit. Recently, it was applied to nanoanchors for protein fixation down to the single molecule level. We now combined STED nanolithography with laser-assisted protein adsorption by photobleaching (LAPAP) for optical and selective attachment of proteins to subdiffractional structures.