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STED Microscopy and Lithography

In 1873, Ernst Abbe has found that resolution in microscopy should be limited to a third of the wavelength [1]. This so called diffraction limit kept its dogmatic character for about 130 years, which is surprising because modern quantum chemistry and quantum optics, which were fully developed at the end of the twenties of the last century, provide basically all necessary ingredients to break this limit. Nevertheless, it took about 65 years until Stefan Hell dared to tackle this dogma and to postulate that taking quantum physics and –optics serious could possibly break Abbe’s diffraction limit [2]. He proposed that the resolution will not be bound to diffraction if one switches off the fluorescence in the outer rim of a diffraction-limited point spread function (PSF) of the excitation beam quick enough so that the fluorophores cannot emit a photon. One possibility (but by far not the only one) to inhibit spontaneous emission is to use STimulated Emission to Deplete (STED) the excited state. In 1999, we experimentally verified the proposal and sub-Abbe resolution was shown in one lateral dimension[3]. One year later, the diffraction barrier was broken in three dimensions [4]. In 2014, Stefan Hell was awarded the Nobel Prize for this invention.

Already in the early papers [3, 4], we pointed out that, in principle, the STED effect is not restricted to increase resolution in confocal fluorescence microscopy, but we proposed that “... this concept can be considered for any application in which the lower excited state of a four-level system is effective. Therefore future applications may well include subdiffraction resolution in pump–probe spectroscopy, three-dimensional photochemistry, and data storage.” [3]

Although very broad (basically covering the whole range of photochemistry), the proposal to improve resolution in any kind of photochemical reaction has up to now only been used to improve resolution in multi-photon lithography [5, 6]. Our own contributions to the state of the art in STED-lithography set the resolution in two dimensions to 120 nm [7] and the feature size in all three dimensions to about 55 nm [6, 7]. Currently, we work on both, an improvement of STED lithography itself but also on applications, specifically by writing nanoscale anchors which allow for bio-functionalization with proteins, down to the single protein level [8, 9].

1. Abbe E.: Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für Mikroskopische Anatomie 1873; 9: 413-68.
2. Hell S. W., Wichmann J.: Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optics Letters 1994; 19(11): 780-2.
3. Klar T. A., Hell S. W.: Subdiffraction resolution in far-field fluorescence microscopy. Optics Letters 1999; 24(14): 954-6.
4. Klar T. A., Jakobs S., Dyba M., Egner A., Hell S. W.: Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proceedings of the National Academy of Sciences of the United States of America 2000; 97(15): 8206-10.
5. Fischer J., Wegener M.: Three-dimensional optical laser lithography beyond the diffraction limit. Laser Photonics Review 2013; 7(1): 22-44.
6. Klar T. A., Wollhofen R., Jacak J.: Sub-Abbe resolution: from STED microscopy to STED lithography. Physica Scripta 2014; T162: 014049.
7. Wollhofen R., Katzmann J., Hrelescu C., Jacak J., Klar T. A.: 120 nm resolution and 55 nm structure size in STED-lithography. Optics Express 2013; 21(9): 10831-40.
8. Wiesbauer M., Wollhofen R., Vasic B., Schilcher K., Jacak J., Klar T. A.: Nano-Anchors with Single Protein Capacity Produced with STED Lithography. Nano Letters 2013; 13(11): 5672-8.
9. Wolfesberger C., Wollhofen R., Buchegger B., Jacak J., Klar T. A.: Streptavidin functionalized polymer nanodots fabricated by visible light lithography. Journal of Nanobiotechnology 2015; 13: 27.

Currently, we are working on the following projects:

** Improvement of STED-Lithography

Supported from core funds, we constantly aim for improving the resolution of STED lithography via both improving the optics as well as improving the chemical components. STED lithography is a quite new field and there is a lot of room for further improvement.

** Three dimensional scaffolds carrying nano-anchors for proteins

The only three dimensional lithography technique which delivers well defined and non-periodic structures is multi-photon polymerization lithography. Given its resolution in the micrometer range, it is well suited for the construction of three dimensional scaffolds that allow for the adhesion and assembly of cells. The development of cellular environments, which are at the same time as defined and as close to nature as possible, is key to ex-vivo experiments on live cells where cell growth, activation, or differentiation are studied. However, no well-defined anchor points for proteins in the nanometer range can be produced using multi-photon lithography. Here, we aim for placing nanoscopic anchor points for proteins via STED lithography inside three dimensional scaffolds.

** Doctorate College Nano Cell

Within the framework of the Doctorate College “Nano Cell”, headed by Prof. Dr. Hinterdorfer from the Biophysics department of JKU, two Doctoral students work in the Institute of Applied Physics on tasks of STED Microscopy and on STED Lithography for molecular dynamics, recognition and organization to membrane transport and motility.