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 (DNAPoints 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.
@article{Kaminska2021,
title = {Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy},
author = {Izabela Kamińska and Johann Bohlen and Renukka Yaadav and Patrick Schüler and Mario Raab and Tim Schröder and Jonas Zähringer and Karolina Zielonka and Stefan Krause and Philip Tinnefeld},
doi = {10.1002/adma.202101099},
year = {2021},
date = {2021-05-01},
urldate = {2021-05-01},
journal = {Advanced Materials},
volume = {33},
number = {24},
pages = {2101099},
publisher = {Wiley},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Masullo2020,
title = {Pulsed Interleaved MINFLUX},
author = {Luciano A. Masullo and Florian Steiner and Jonas Zähringer and Lucía F. Lopez and Johann Bohlen and Lars Richter and Fiona Cole and Philip Tinnefeld and Fernando D. Stefani},
doi = {10.1021/acs.nanolett.0c04600},
year = {2020},
date = {2020-12-01},
journal = {Nano Letters},
volume = {21},
number = {1},
pages = {840--846},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Kaminska2019,
title = {Distance Dependence of Single-Molecule Energy Transfer to Graphene Measured with DNA Origami Nanopositioners},
author = {I. Kaminska and J. Bohlen and S. Rocchetti and F. Selbach and G. P. Acuna and P. Tinnefeld},
doi = {10.1021/acs.nanolett.9b00172},
year = {2019},
date = {2019-06-01},
journal = {Nano Letters},
volume = {19},
number = {7},
pages = {4257--4262},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Isbaner2018,
title = {Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer},
author = {Sebastian Isbaner and Narain Karedla and Izabela Kaminska and Daja Ruhlandt and Mario Raab and Johann Bohlen and Alexey Chizhik and Ingo Gregor and Philip Tinnefeld and Jörg Enderlein and Roman Tsukanov},
doi = {10.1021/acs.nanolett.8b00425},
year = {2018},
date = {2018-03-01},
journal = {Nano Letters},
volume = {18},
number = {4},
pages = {2616--2622},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Raab2017,
title = {Shifting molecular localization by plasmonic coupling in a single-molecule mirage},
author = {Mario Raab and Carolin Vietz and Fernando Daniel Stefani and Guillermo Pedro Acuna and Philip Tinnefeld},
doi = {10.1038/ncomms13966},
year = {2017},
date = {2017-01-01},
journal = {Nature Communications},
volume = {8},
number = {1},
publisher = {Springer Science and Business Media LLC},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Schmied_2012,
title = {Fluorescence and super-resolution standards based on DNA origami},
author = {Jürgen J Schmied and Andreas Gietl and Phil Holzmeister and Carsten Forthmann and Christian Steinhauer and Thorben Dammeyer and Philip Tinnefeld},
doi = {10.1038/nmeth.2254},
year = {2012},
date = {2012-12-01},
journal = {Nature Methods},
volume = {9},
number = {12},
pages = {1133--1134},
publisher = {Springer Science and Business Media LLC},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Jungmann_2010,
title = {Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami},
author = {Ralf Jungmann and Christian Steinhauer and Max Scheible and Anton Kuzyk and Philip Tinnefeld and Friedrich C. Simmel},
doi = {10.1021/nl103427w},
year = {2010},
date = {2010-10-01},
journal = {Nano Letters},
volume = {10},
number = {11},
pages = {4756--4761},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Steinhauer_2009,
title = {DNA Origami as a Nanoscopic Ruler for Super-Resolution Microscopy},
author = {Christian Steinhauer and Ralf Jungmann and Thomas L. Sobey and Friedrich C. Simmel and Philip Tinnefeld},
doi = {10.1002/anie.200903308},
year = {2009},
date = {2009-11-01},
urldate = {2009-11-01},
journal = {Angewandte Chemie International Edition},
volume = {48},
number = {47},
pages = {8870--8873},
publisher = {Wiley},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Heilemann_2008,
title = {Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes},
author = {Mike Heilemann and Sebastian vanhspace0.25emdehspace0.25emLinde and Mark Schüttpelz and Robert Kasper and Britta Seefeldt and Anindita Mukherjee and Philip Tinnefeld and Markus Sauer},
doi = {10.1002/anie.200802376},
year = {2008},
date = {2008-08-01},
journal = {Angewandte Chemie International Edition},
volume = {47},
number = {33},
pages = {6172--6176},
publisher = {Wiley},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
@article{Heilemann_2005,
title = {Carbocyanine Dyes as Efficient Reversible Single-Molecule Optical Switch},
author = {Mike Heilemann and Emmanuel Margeat and Robert Kasper and Markus Sauer and Philip Tinnefeld},
doi = {10.1021/ja044686x},
year = {2005},
date = {2005-02-01},
journal = {Journal of the American Chemical Society},
volume = {127},
number = {11},
pages = {3801--3806},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
