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Nanoscopic Ruler

The improvement of optical microscopy towards higher resolution has been subject to extensive research over the past years. The diffraction limit of light prohibits resolving details smaller than half its wavelength, resulting in a fundamental resolution limit of 250-300 nm. It is the goal, however, to reach a resolution that enables imaging of much smaller, closely packed structures by optical microscopy. Exciting developments in fluorescence microscopy have enabled far-field imaging beyond this diffraction limit. Several approaches have been realized within the last years yielding fluorescence images of unprecedented sharpness and resolution. One such method that uses the subsequent localization of single fluorescent molecules has been developed by the group of Prof. Tinnefeld at the LMU. For further development of the field, structures with defined numbers of fluorescent molecules at precisely determined positions are required that allow comparison and evaluation of the approaches as well as calibration of each superresolution fluorescence microscope. In this context, a nano-scale ruler would be very much appreciated.

Independent of the developments of optical microscopy, physicists from the group of Prof. Simmel at the Technical University of Munich have advanced the use of self-assembled DNA nanostructures. In the so-called "DNA origami technique", single stranded DNA molecules can be made to self-assemble into well-defined two-dimensional macromolecular structures. The name "origami" comes from the fact that a long single stranded DNA molecule is "folded" into a particular shape. With a diameter of typically 100 nm and a fully addressable breadboard-like structure, rectangular DNA origami structures can be used to arrange nanoscale objects with nanometer precision. This very feature makes DNA origami the ideal sample for super-resolution microscopy.


Picture:
© Angew Chem Int Ed 48, 8870-8873 (2009)

Figure: TIRF image of surface-immobilized DNA origami containing two ATTO655-labeled staple strands. The positions of the single fluorophores cannot be determined because of their overlapping pointspread functions. b) Super-resolution image of the same region using blink microscopy: Single fluorophore positions are clearly resolved. Scale bar: 500nm.

 

Brought together by the International Doctorate Program NanoBioTechnology, PhD students Tom Sobey and Christian Steinhauer shared their expertise to create a nanoscopic ruler made of DNA. They showed that different super-resolution techniques can be used to resolve the distance between two fluorophores grafted into the DNA origami structure. The theoretically designed distance was not only measured experimentally with a deviation of only one nanometer, but it could also be shown that blink microscopy is capable of optically resolving 90 % of the structures. This demonstrates the robustness of both DNA origami as nanoscopic ruler as well as blink microscopy, thus offering the required calibration structures for superresolution microscopies.

 

Publication:
"DNA Origami as Nanoscopic Ruler for Superresolution Microscopy"
C. Steinhauer, R. Jungmann, T. L. Sobey, F. C. Simmel, P. Tinnefeld
Angew Chem Int Ed 48, 8870-8873 (2009)

 

Tom Sobey
Education

since 2006
PhD student in the group of
Prof. Friedrich Simmel, TUM

2004 - 2005
Research Assistant, Centre for Quantum Computer Technology,
Sydney

1999 - 2004
Bachelor of Science in Physics with First Class Honours

Selected Publication

T. L. Sobey, S. Renner, F. C. Simmel:  
"Assembly and melting of DNA nanotubes from single-sequence tiles"
Journal of Physics Condensed Matter,  21,  034112 (2009)

Christian Steinhauer
Education

since 2007
PhD student in the group of
Prof. Philip Tinnefeld, now TU Braunschweig

2002 - 2007
Studies in Molecular Biotechnology at the University of Bielefeld and the University of Adelaide

Selected Publication

B. Person, I. H. Stein, C. Steinhauer, J. Vogelsang and P. Tinnefeld:
"Correlated movement and bending of nucleotide acid structures visualized by multicolor single-molecule spectroscopy"
ChemPhysChem, 10:1455 (2009)