Richard Wollhofen
Johannes Kepler University of Linz
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Publication
Featured researches published by Richard Wollhofen.
Optics Express | 2013
Richard Wollhofen; Julia Katzmann; Calin Hrelescu; Jaroslaw Jacak; Thomas A. Klar
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.
Physica Scripta | 2014
Thomas A. Klar; Richard Wollhofen; Jaroslaw Jacak
We review the emergence of three-dimensional sub-Abbe optical nanoscopy, which in its original version deployed stimulated-emission-induced depletion (STED) of the excited state of the fluorophores to break the diffraction limit. We focus first on different methods to realize a donut-shaped point spread function (PSF), which are key to distribute the intensity of the depleting beam around the originally excited focal volume, and second on methods to suppress fluorescence, which are alternative to stimulated emission. In the second part, we elaborate on the transition of STED microscopy to STED lithography. We present data which show the state of the art of minimal axial feature sizes (around 55 nm). Again, stimulated emission is not the only technique to decrease the feature size and the resolution in optical lithography. Alternative techniques are up-conversion within the triplet system or excitation of polymerization stoppers inside the donut-shaped depletion PSF. We conclude with a comparison of the record resolutions in STED and STED-related lithography techniques, which are in the range of some tens of nanometers, far below the wavelength of visible light.
Nano Letters | 2013
Moritz Wiesbauer; Richard Wollhofen; Borislav Vasić; Kurt Schilcher; Jaroslaw Jacak; Thomas A. Klar
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.
ACS Nano | 2016
Bianca Buchegger; Johannes Kreutzer; Birgit Plochberger; Richard Wollhofen; Dmitry Sivun; Jaroslaw Jacak; Gerhard J. Schütz; Ulrich S. Schubert; Thomas A. Klar
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.
Royal Society Open Science | 2017
Cristina Plamadeala; Florian Hischen; R. Friesenecker; Richard Wollhofen; Jaroslaw Jacak; Gerda Buchberger; Ernst Heiss; Thomas A. Klar; Werner Baumgartner; J. Heitz
Nature has always served as an inspiration for scientists, helping them to solve a large diversity of technical problems. In our case, we are interested in the directional transport of oily liquids and as a model for this application we used the flat bug Dysodius lunatus. In this report, we present arrays of drops looking like polymer microstructures produced by the two-photon polymerization technique that mimic the micro-ornamentation from the bugs cuticle. A good directionality of oil transport was achieved, directly controlled by the direction of the pointed microstructures at the surface. If the tips of the drop-like microstructures are pointing towards the left side, the liquid front moves to the right and vice versa. Similar effects could be expected for the transport of oily lubricants. These results could, therefore, be interesting for applications in friction and wear reduction.
Journal of Biomedical Materials Research Part A | 2017
J. Heitz; Cristina Plamadeala; Moritz Wiesbauer; Peter Freudenthaler; Richard Wollhofen; Jaroslaw Jacak; Thomas A. Klar; Birte Magnus; D Kostner; Agnes Weth; Werner Baumgartner; Rainer Marksteiner
Abstract The main aim of this work was to stimulate bone‐forming cells to produce three‐dimensional networks of mineralized proteins such as those occurring in bones. This was achieved by a novel approach using a specific type of mesenchymal progenitor cells (i.e., primary fibroblast cells from human hair roots) seeded on to polymer scaffolds. We wrote polymer microstructures with one or more levels of quadratic pores on to a flexible substrate by means of two‐photon polymerization using a Ti‐sapphire femtosecond laser focused into a liquid acrylate‐based resin containing a photoinitiator. Progenitor cells, differentiated into an osteogenic lineage by the use of medium supplemented with biochemical stimuli, can be seeded on to the hydrophilic three‐dimensional scaffolds. Due to confinement to the microstructures and/or mechanical interaction with the scaffold, the cells are stimulated to produce high amounts of calcium‐binding proteins, such as collagen type I, and show an increased activation of the actin cytoskeleton. The best results were obtained for quadratic pore sizes of 35 µm: the pore volumes become almost filled with both cells in close contact with the walls of the structure and with extracellular matrix material produced by the cells.
Journal of Nanobiotechnology | 2015
Clemens Wolfesberger; Richard Wollhofen; Bianca Buchegger; Jaroslaw Jacak; Thomas A. Klar
BackgroundTwo-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.ResultsHere 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.ConclusionThe 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.
ACS Applied Materials & Interfaces | 2018
Richard Wollhofen; Markus Axmann; Peter Freudenthaler; Christian Gabriel; Clemens Röhrl; Herbert Stangl; Thomas A. Klar; Jaroslaw Jacak
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.
Analytical Chemistry | 2018
Bianca Buchegger; Johannes Kreutzer; Markus Axmann; Sandra Mayr; Richard Wollhofen; Birgit Plochberger; Jaroslaw Jacak; Thomas A. Klar
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.
european quantum electronics conference | 2017
Bianca Buchegger; Johannes Kreutzer; Sandra Mayr; Richard Wollhofen; Jaroslaw Jacak; Thomas A. Klar
Two-Photon Polymerization enables fabrication of two and three dimensional polymer structures. For structuring, a highly cross-linking acrylate monomer is mixed with a photo-initiator. Using femtosecond IR light as an excitation source, feature sizes down to 100 nm can be achieved. Applying stimulated emission depletion (STED) reduces the feature size even further [1, 2]. Recently, our group developed functional polymer structures below the diffraction limit by mixing metal oxo clusters into the acrylate-based photoresist [3].