Single-Molecule Four-Color FRET
Due to the complexity of these supra-molecular constructs we expected considerable sample heterogeneity. This heterogeneity is best addressed using single-molecule techniques. For the FRET results reported above, a new single-molecule four-color FRET technique with alternating laser excitation that allows studying the interaction between six energy transfer pairs quasi-simultaneously was developed. The setup is based on an inverted confocal microscope and uses the combination of a supercontinuum laser for free wavelength selection with acousto-optical filters (AOTFs) and beamsplitter (AOBS) for excitation alternation and optimized detection sensitivity on all channels. Alternating laser excitation enables direct probing of the stoichiometry by subsequently exciting all four fluorophores. The different subpopulations are evaluated by the relative intensities in so-called stoichiometry-histograms (fig. 4).
In the two-dimensional histogram comparing all four dyes in the construct, the population around 0.5 on both axes represents intact DNA origami with all four fluorophores. Interestingly, detailed analysis revealed that origami structures were formed with very high yield and sample heterogeneity mainly originated from inactive subpopulations of the IR-dye. Energy transfer was analyzed one molecule at a time and the results were plotted in histograms (fig. 3). Detailed analysis revealed that for the selected thresholds for > 98% of the molecules, the position of the jumper dye could be correctly assigned.
Yardsticks for Super-Resolution Microscopy
Positioning dyes at distances too large for FRET to occur is the key for one of the first practical applications of DNA nanostructures, that is, DNA nano-scale yardsticks for super-resolution microscopy.
The common principle of super-resolution microscopy by subsequent single-molecule localizations is that only one fluorophore is active for a diffraction-limited area at any given time. Every fluorophore is localized by imaging with a sensitive camera and the molecules' positions are summed up in histograms to obtain the reconstructed super-resolved images.
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To compare super-resolution techniques and to test super-resolution microscopes, we developed microscopy standards with an exactly defined number of fluorophores at programmed distances in the range of 10-200 nm prepared by the DNA origami technique . We used different super-resolution techniques such as Blink-Microscopy  which exploits radical ion dark states, STED, DNA-PAINT , and dSTORM  to resolve distances between single molecules or lines of molecules beyond the diffraction limit. In figure 5, an example of two dyes separated by 60 nm resolved by DNA-PAINT is depicted. These nanoscopic yardsticks can be easily distributed and might become an important alignment tool for super-resolution microscopes.
In summary, the combination of DNA as a template with photonic functionalities placed on top is becoming an emerging field of research and might have the potential to fill the gap between top-down lithography and bottom-up self-assembly.
The authors are grateful to Sebastian Laurien and Christian Steinhauer for their help with the manuscript and figures. The work was supported by the DFG and the excellence cluster Nanosystems Initiative Munich (NIM).
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Prof. Dr. Philip Tinnefeld (corresponding author)
Institute of physical and theoretical Chemistry, Braunschweig University of Technology, Germany
& Center for NanoScience, Munich, Germany
Applied Physics - Biophysics & Center for NanoScience
Ludwig Maximilian University, Munich, Germany
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