Two–photon direct laser writing (DLW) lithography is limited in the achievable structure size as well as in structure resolution. Adding stimulated emission depletion (STED) to DLW allowed overcoming both restrictions. We now push both to new limits. Using visible light for two-photon DLW (780 nm) and STED (532 nm), we obtain lateral structure sizes of 55 nm, a Sparrow limit of around 100 nm and we present two clearly separated lines spaced only 120 nm apart. The photo-resist used in these experiments is a mixture of tri- and tetra-acrylates and 7-Diethylamino-3-thenoylcoumarin as a photo-starter which can be readily quenched via STED.

Alternative splicing (AS) is an important regulatory process that leads to the creation of multiple RNA transcripts from a single gene. Alternative transcripts often carry premature termination codons (PTCs), which trigger nonsense-mediated decay (NMD), a cytoplasmic RNA degradation pathway. However, intron retention, the most prevalent AS event in plants, often leads to PTC-carrying splice variants that are insensitive to NMD; this led us to question the fate of these special RNA variants. Here, we present an innovative approach to monitor and characterize endogenous mRNA splice variants within living plant cells. This method combines standard confocal laser scanning microscopy for molecular beacon detection with a robust statistical pipeline for sample comparison. We demonstrate this technique on the localization of NMD-insensitive splice variants of two Arabidopsis thaliana genes, RS2Z33 and the SEF factor. The experiments reveal that these intron-containing splice variants remain within the nucleus, which allows them to escape the NMD machinery. Moreover, fluorescence recovery after photobleaching experiments in the nucleoplasm show a decreased mobility of intron-retained mRNAs compared with fully spliced RNAs. In addition, differences in mobility were observed for an mRNA dependent on its origin from an intron-free or an intron-containing gene.

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro 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. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood–brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

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. The depletion efficiency in dependence of the cluster loading was investigated and full depletion of the STED effect was observed with a cluster loading exceeding 4 wt %. A feature size by λ/11 was achieved by using a donut-shaped depletion beam. The reactivity of the mercapto groups on the surface of the nanostructure was tested by incubation with mercapto-reactive fluorophores.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

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 via 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. Assessment of biocompatibility of the new resins was done by cultivating human umbilical vein endothelial cells on two-dimensionally structured substrates for four days. The cell density and presence of apoptotic cells has been quantified.