Visualization 3: Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

Dragging of an indiretcly trapped cell with a two-photon polymerized cell manipulator perpendicular to its symmetry axis. Originally published in Biomedical Optics Express on 01 January 2016 (boe-7-1-45

Visualization 2: Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

Dragging of an indiretcly trapped cell with a two-photon polymerized cell manipulator along its symmetry axis. Originally published in Biomedical Optics Express on 01 January 2016 (boe-7-1-45

Nano-Anchors with Single Protein Capacity Produced with STED Lithography

Acrylate nanoanchors of subdiffraction-limited diameter are written with optical stimulated emission depletion (STED) lithography. After incubation, 98% of all nanoanchors are loaded quickly with fluorescently labeled antibodies. Controlling the size of the nanoanchors allows for limiting the number of the antibodies. Direct stochastic optical reconstruction microscopy (dSTORM) imaging, statistical distribution of fluorescence, quantitative fluorescence readout, and single molecule blinking consistently prove that 80% of the nanoanchors with a 65 nm diameter are carrying only one antibody each, which are functional as confirmed with live erythrocytes

Nano-Anchors with Single Protein Capacity Produced with STED Lithography

Acrylate nanoanchors of subdiffraction-limited diameter are written with optical stimulated emission depletion (STED) lithography. After incubation, 98% of all nanoanchors are loaded quickly with fluorescently labeled antibodies. Controlling the size of the nanoanchors allows for limiting the number of the antibodies. Direct stochastic optical reconstruction microscopy (dSTORM) imaging, statistical distribution of fluorescence, quantitative fluorescence readout, and single molecule blinking consistently prove that 80% of the nanoanchors with a 65 nm diameter are carrying only one antibody each, which are functional as confirmed with live erythrocytes

Visualization 1: Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

Sequence of indirect optical manipulation of a single cell showing the trapping of a polymerized cross, its rotation, cross-cell attachment and cell translation. Originally published in Biomedical Optics Express on 01 January 2016 (boe-7-1-45

Multiphoton-Polymerized 3D Protein Assay

Multiphoton polymerization (MPP) enables 3D fabrication of micro- and nanoscale devices with complex geometries. Using MPP, we create a 3D platform for protein assays. Elevating the protein-binding sites above the substrate surface allows an optically sectioned readout, minimizing the inevitable background signal from nonspecific protein adsorption at the substrate surface. Two fluorescence-linked immunosorbent assays are demonstrated, the first one relying on streptavidin–biotin recognition and the second one on antibody recognition of apolipoprotein A1, a major constituent of high-density lipoprotein particles. Signal-to-noise ratios exceeding 1000 were achieved. The platform has high potential for 3D multiplexed recognition assays with an increased binding surface for on-chip flow cells

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. 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

Biomolecular Characterization of Putative Antidiabetic Herbal Extracts

<div><p>Induction of GLUT4 translocation in the absence of insulin is considered a key concept to decrease elevated blood glucose levels in diabetics. Due to the lack of pharmaceuticals that specifically increase the uptake of glucose from the blood circuit, application of natural compounds might be an alternative strategy. However, the effects and mechanisms of action remain unknown for many of those substances. For this study we investigated extracts prepared from seven different plants, which have been reported to exhibit anti-diabetic effects, for their GLUT4 translocation inducing properties. Quantitation of GLUT4 translocation was determined by total internal reflection fluorescence (TIRF) microscopy in insulin sensitive CHO-K1 cells and adipocytes. Two extracts prepared from purslane (<i>Portulaca oleracea</i>) and tindora (<i>Coccinia grandis</i>) were found to induce GLUT4 translocation, accompanied by an increase of intracellular glucose concentrations. Our results indicate that the PI3K pathway is mainly responsible for the respective translocation process. Atomic force microscopy was used to prove complete plasma membrane insertion. Furthermore, this approach suggested a compound mediated distribution of GLUT4 molecules in the plasma membrane similar to insulin stimulated conditions. Utilizing a fluorescent actin marker, TIRF measurements indicated an impact of purslane and tindora on actin remodeling as observed in insulin treated cells. Finally, <i>in-ovo</i> experiments suggested a significant reduction of blood glucose levels under tindora and purslane treated conditions in a living organism. In conclusion, this study confirms the anti-diabetic properties of tindora and purslane, which stimulate GLUT4 translocation in an insulin-like manner.</p></div

Quantitation of GLUT4 translocation and glucose uptake in 3T3-L1 cells.

<p>(A) TIRF microscopy based quantitation of GLUT4 translocation in 3T3-L1 GLUT4-GFP adipocytes. Cells were grown in 96-well plates (20,000 cells/well) for 24 hours and then starved overnight in serum-free medium. Images before and 10 minutes after stimulation with indicated substances are shown. Scale bar = 20 μm. (B) GLUT4-GFP signal intensity increase was analyzed after 10 and 30 minutes of stimulation. Error bars are based on the standard error of the mean. **P < 0.01, ***P < 0.001 and ****P < 0.0001, significant increase with respect to KRPH treated cells. (C) For glucose uptake measurements 3T3-L1 cells were grown in 96-well plates (2,000 cells/well) and differentiated to adipocytes. Cells were starved in serum-free medium overnight, glucose deprived in KRPH buffer for 40 minutes, and then stimulated with the indicated substances for 20 minutes followed by addition of 2-DG (20 minutes). Cell extracts were prepared and 2-DG uptake was measured after a colorimetric reaction using a plate reader device. Samples were measured in duplicates at least in three individual experiments. Error bars are based on the standard error of the mean. *P < 0.05, **P < 0.01 and ****P < 0.0001, significant increase with respect to KRPH (Ins, PP60, PUR) or 0.25% DMSO in KRPH (TIN, GIN, BIL, JIA, MT) treated cells.</p

HET-CAM experiments for the determination of blood glucose decreasing properties of selected herbal compounds <i>in-ovo</i>.

<p>(A) The chorioallantoic membrane of chicken embryos was incubated with the tested substances for 3 hours. After removal of the egg membrane (1), blood was collected from a main vessel (2), and the concentration of glucose in prepared plasma samples was measured by HPLC. (B) Normalized glucose concentration after incubation at indicated time points. Error bars are based on the standard error of the mean. *P < 0.05, ***P < 0.001 and ****P < 0.0001, significant decrease with respect to KRPH (NovoRapid, PUR) or DMSO (TIN) treated cells.</p