New buffer systems for photopainting of single biomolecules.

We present newly developed buffer systems that significantly improve the efficiency of a photochemically induced surface modification at the single molecule level. Buffers with paramagnetic cations and radical oxygen promoting species facilitate laser-assisted protein adsorption by photobleaching (LAPAP) of single fluorescently labelled oligonucleotides or biotin onto multi-photon-lithography-structured 2D and 3D acrylate scaffolds. Single molecule fluorescence microscopy has been used to quantify photopainting efficiency.

Multiphoton lithography with protein photoresists.

Recently, 2D/3D direct laser writing has attracted increased attention due to its broad applications ranging from biomedical engineering to aerospace. 3D nanolithography of water-soluble protein-based scaffolds have been envisioned to provide a variety of tunable properties. In this paper, we present a functional protein-based photoresist with tunable mechanical properties that is suitable for multiphoton lithography (MPL).

Low-Fluorescence Starter for Optical 3D Lithography of Sub-40 nm Structures.

Stimulated emission depletion (STED) has been used to break the diffraction limit in fluorescence microscopy. Inspired by this success, similar methods were used to reduce the structure size in three-dimensional, subdiffractional optical lithography. So far, only a very limited number of radical polymerization starters proved to be suitable for STED-inspired lithography.

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.

Label-free characterization of an extracellular vesicle-based therapeutic.

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements.

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer.

Dual Channel Microfluidics for Mimicking the Blood-Brain Barrier.

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance.

Plasmon-Assisted Direction- and Polarization-Sensitive Organic Thin-Film Detector.

Utilizing Bragg surface plasmon polaritons (SPPs) on metal nanostructures for the use in optical devices has been intensively investigated in recent years. Here, we demonstrate the integration of nanostructured metal electrodes into an ITO-free thin film bulk heterojunction organic solar cell, by direct fabrication on a nanoimprinted substrate. The nanostructured device shows interesting optical and electrical behavior, depending on angle and polarization of incidence and the side of excitation.

3D multiphoton lithography using biocompatible polymers with specific mechanical properties.

The fabrication of two- and three-dimensional scaffolds mimicking the extracellular matrix and providing cell stimulation is of high importance in biology and material science. We show two new, biocompatible polymers, which can be 3D structured multiphoton lithography, and determine their mechanical properties. Atomic force microscopy analysis of structures with sub-micron feature sizes reveals Young's modulus values in the 100 MPa range.

Optical Coulomb blockade lifting in plasmonic nanoparticle dimers.

If two metal nanoparticles are ultimately approached, a tunneling current prevents both an infinite redshift of the bonding dipolar plasmon and an infinite increase of the electric field in the hot spot between the nanoparticles. We argue that a Coulomb blockade suppresses the tunneling current and sustains a redshift even for sub-nanometer approach up to moderate fields. Only for stronger optical fields, the Coulomb blockade is lifted and a charge transfer plasmon is formed.

Plasmonic Horizon in Gold Nanosponges.

An electromagnetic wave impinging on a gold nanosponge coherently excites many electromagnetic hot-spots inside the nanosponge, yielding a polarization-dependent scattering spectrum. In contrast, a hole, recombining with an electron, can locally excite plasmonic hot-spots only within a horizon given by the lifetime of localized plasmons and the speed carrying the information that a plasmon has been created. This horizon is about 57 nm, decreasing with increasing size of the nanosponge.

Hybrid Multilayered Plasmonic Nanostars for Coherent Random Lasing.

Here, we report that hybrid multilayered plasmonic nanostars can be universally used as feedback agents for coherent random lasing in polar or nonpolar solutions containing gain material. We show that silver-enhancement of gold nanostars reduces the pumping threshold for coherent random lasing substantially for both a typical dye (R6G) and a typical fluorescent polymer (MEH-PPV). Further, we reveal that the lasing intensity and pumping threshold of random lasers based on silver-enhanced gold nanostars are not influenced by the silica coating, in contrast to gold nanostar-based random lasers, where silica-coated gold nanostars support only amplified spontaneous emission but no coherent random lasing.

Anticorrelation of Photoluminescence from Gold Nanoparticle Dimers with Hot-Spot Intensity.

Bulk gold shows photoluminescence (PL) with a negligible quantum yield of ∼10, which can be increased by orders of magnitude in the case of gold nanoparticles. This bears huge potential to use noble metal nanoparticles as fluorescent and unbleachable stains in bioimaging or for optical data storage. Commonly, the enhancement of the PL yield is attributed to nanoparticle plasmons, specifically to the enhancements of scattering or absorption cross sections.

Stimulated Emission Depletion Lithography with Mercapto-Functional Polymers.

Surface reactive nanostructures were fabricated using stimulated emission depletion (STED) lithography. The functionalization of the nanostructures was realized by copolymerization of a bifunctional metal oxo cluster in the presence of a triacrylate monomer. Ligands of the cluster surface cross-link to the monomer during the lithographic process, whereas unreacted mercapto functionalized ligands are transferred to the polymer and remain reactive after polymer formation of the surface of the nanostructure.