Superresolution microscopy

While conventional fluorescence microscopy is one of the most used techniques to study biological systems and different type of materials, it is intrinsically limited in spatial resolution to 200-300 nm due to the diffraction of light. Super-resolution microscopy has overcome this limitation by exploiting the controlled switching of fluorescent dyes between bright (emitting) and dark (not emitting) states, enabling a 10 to 100-fold resolution enhancement. This improvement in spatial resolution represented a breakthrough in life and materials science, since it allowed to directly visualize how molecules are organized at the nanometer scale.

A major group of super-resolution techniques involves the localization of sparse single emitters with sub-diffraction precision. This family of methods is called Single Molecule Localization Microscopy (SMLM), and our lab is involved in the development and application of such techniques already from its early days. Our most prominent examples are the inventions of direct Stochastic Optical Reconstruction Microscopy (dSTORM) in cooperation with the Sauer group (Würzburg University), and DNA-PAINT (DNA Points Accumulation for Imaging in Nanoscale Topography) together with the Simmel group (TU Munich). Both techniques are nowadays workhorses in biophysical research: their use has shed light into many opened questions in different fields, including the discovery of new supramolecular biological structures. 

One big challenge in the field of super-resolution microscopy consists of the development of techniques capable of reaching both high temporal and spatial resolution. Towards this direction, we recently developed pulsed interleaved-MINFLUX in collaboration with the Stefani group (CIBION-Conicet). This technique enables imaging and single molecule tracking with molecular scale resolution (~1 nm), while its temporal resolution is only limited by the repetition rate of the laser pulse (~MHz range). In addition, p-MINFLUX provides single-molecule fluorescence lifetime information, which is key to study the conformation and evolution of the local environment of the single emitters.

Moreover, we have exploited the distance dependent energy transfer from single molecules to gold (MIET: Metal-Induced Energy Transfer) or graphene (GET: Graphene Energy Transfer) in order to achieve nanometer resolution also in axial localization, thus completing the toolkit to perform super-resolution microscopy in all three dimensions with nanometer resolution.

Figure 1. Top left: DNA-PAINT principle. The on-off switching is obtained by the transient hybridization of single-stranded DNA labelled with fluorescent dyes (called ‘imager’ strands) to single-stranded DNA bound to the target (called ‘docking’ strands) (Jungmann, R. et al., 2010). Top right: p-MINFLUX principle. Four doughnut-shaped excitation foci are used to interrogate the position of a single emitter. In this technique, the four beams are obtained by splitting a single pulsed laser beam into four different branches which are shifted in time in the nanoseconds scale (Masullo, L. et al., 2020). Bottom: Graphene Energy Transfer principle. The energy transfer process follows a d-4 power law, which allows to resolve the axial position of single molecules with nanometer resolution. In combination with DNA-PAINT, it enables sub-10 nm resolution in all three dimensions (Kamińska, I. et al., 2021).

In addition to the development of new methods, we have a deep focus on applying those techniques in the field of DNA nanotechnology. We routinely use DNA origami structures as a platform to place fluorescent dyes at predesigned positions with unique control. From this point of view, DNA structures act as a sample breadboard of superb flexibility. Most fundamentally, we invented fluorescent nanorulers for single molecule localization microscopy and other techniques, where dyes can be placed in a controlled manner at distances ranging between 6 and 400 nm. These nanorulers represent the very first commercial DNA origami product, that is sold by our spin-off company GATTAquant. Moreover, they are the experimental basis for numerous exciting, scientific experiments, like the spatially resolved investigation of plasmonic nanoantennas or the benchmarking of new super-resolution methods, among others.


Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy

Kamińska, Izabela; Bohlen, Johann; Yaadav, Renukka; Schüler, Patrick; Raab, Mario; Schröder, Tim; Zähringer, Jonas; Zielonka, Karolina; Krause, Stefan; Tinnefeld, Philip

Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy Journal Article

In: Advanced Materials, vol. 33, no. 24, pp. 2101099, 2021.

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Masullo, Luciano A.; Steiner, Florian; Zähringer, Jonas; Lopez, Lucía F.; Bohlen, Johann; Richter, Lars; Cole, Fiona; Tinnefeld, Philip; Stefani, Fernando D.

Pulsed Interleaved MINFLUX Journal Article

In: Nano Letters, vol. 21, no. 1, pp. 840–846, 2020.

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Kaminska, I.; Bohlen, J.; Rocchetti, S.; Selbach, F.; Acuna, G. P.; Tinnefeld, P.

Distance Dependence of Single-Molecule Energy Transfer to Graphene Measured with DNA Origami Nanopositioners Journal Article

In: Nano Letters, vol. 19, no. 7, pp. 4257–4262, 2019.

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Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer Journal Article

In: Nano Letters, vol. 18, no. 4, pp. 2616–2622, 2018.

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Raab, Mario; Vietz, Carolin; Stefani, Fernando Daniel; Acuna, Guillermo Pedro; Tinnefeld, Philip

Shifting molecular localization by plasmonic coupling in a single-molecule mirage Journal Article

In: Nature Communications, vol. 8, no. 1, 2017.

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Schmied, Jürgen J; Gietl, Andreas; Holzmeister, Phil; Forthmann, Carsten; Steinhauer, Christian; Dammeyer, Thorben; Tinnefeld, Philip

Fluorescence and super-resolution standards based on DNA origami Journal Article

In: Nature Methods, vol. 9, no. 12, pp. 1133–1134, 2012.

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Jungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C.

Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami Journal Article

In: Nano Letters, vol. 10, no. 11, pp. 4756–4761, 2010.

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Steinhauer, Christian; Jungmann, Ralf; Sobey, Thomas���L.; Simmel, Friedrich���C.; Tinnefeld, Philip

DNA Origami as a Nanoscopic Ruler for Super-Resolution Microscopy Journal Article

In: Angewandte Chemie International Edition, vol. 48, no. 47, pp. 8870–8873, 2009.

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Heilemann, Mike; vanhspace0.25emdehspace0.25emLinde, Sebastian; Schüttpelz, Mark; Kasper, Robert; Seefeldt, Britta; Mukherjee, Anindita; Tinnefeld, Philip; Sauer, Markus

Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes Journal Article

In: Angewandte Chemie International Edition, vol. 47, no. 33, pp. 6172–6176, 2008.

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Heilemann, Mike; Margeat, Emmanuel; Kasper, Robert; Sauer, Markus; Tinnefeld, Philip

Carbocyanine Dyes as Efficient Reversible Single-Molecule Optical Switch Journal Article

In: Journal of the American Chemical Society, vol. 127, no. 11, pp. 3801–3806, 2005.

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