STED lithography in microfluidics for 3D thrombocyte aggregation testing.

Three-dimensional photopolymerization techniques such as multiphoton polymerization lithography (MPL) and stimulated emission depletion (STED) lithography are powerful tools for fabricating structures in the sub-µm range. Combining these techniques with microfluidics enables us to broaden the range of their applications. In this study, we show a microfluidic device enhanced with MPL structures carrying STED-lithographically written nanoanchors that promote binding of the von Willebrand factor (vWF). The density of vWF is adjusted by varying the number of the nanoanchors on the 3D structures. This allows us to study the impact of the density of vWF on the activation of thrombocytes. The activation of the thrombocytes seems to decrease with the density of vWF on the 3D scaffolds inside the microfluidic channels.

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 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.

Proteins on Supported Lipid Bilayers Diffusing around Proteins Fixed on Acrylate Anchors.

Mobility of proteins and lipids plays a major role in physiological processes. Platforms which were developed to study protein interaction between immobilized and mobile proteins suffer from shortcomings such as fluorescence quenching or complicated fabrication methods. Here we report a versatile platform comprising immobilized histidine-tagged proteins and biotinylated proteins in a mobile phase. Importantly, multiphoton photolithography was used for easy and fast fabrication of the platform and allows, in principle, extension of its application to three dimensions. The platform, which is made up of functionalized polymer structures embedded in a mobile lipid bilayer, shows low background fluorescence and allows for mobility of arbitrary proteins.

Frequency domain photoacoustic and fluorescence microscopy.

We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain.

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.

Streptavidin functionalized polymer nanodots fabricated by visible light lithography.

BACKGROUND: Two-photon polymerization, optionally combined with stimulated emission depletion (STED) lithography, allows two and three dimensional polymer fabrication with structure sizes and resolution below the diffraction limit. Structuring of polymers with photons, whose wavelength is within the visible range of the electromagnetic spectrum, gives new opportunities to a large field of applications e.g. in the field of biotechnology and tissue engineering. In order to create new biotechnological applications, versatile methods are needed to functionalize the polymeric structures. RESULTS: Here we report the creation of polymer-nanodots with high streptavidin (SA) affinity via two-photon polymerization (TPP). Controlling the size of the polymer dots allows for limiting the number of the SA molecules. TPP dots with a diameter of a few 100 nm show up to 100% streptavidin loading. We can show that most of the dots are loaded by one to two streptavidins on average. Attached streptavidin molecules remain functional and are capable to bind 0.7 biotin molecules on average. CONCLUSION: The presented functionalized nanostructures may be used as platforms for a multitude of biological experimental setups. Nanoscopic well defined structures, capable of selective binding of streptavin proteins, used as linkers for other biotinylated biomolecules, may also find application in in-vitro sensing, like for example lab on chip devices with limited surface area.