124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics.

Orsolya K Wade,Heinrich Grabmayr,Philipp C Nickels,Mahipal Ganji,Sebastian Strauss,Maximilian T Strauss,Joerg Schnitzbauer,Peng Yin,Petra Schwille,Florian Stehr,Florian Schueder,Ralf Jungmann,Johannes Stein,Johannes B Woehrstein

doi:10.1021/acs.nanolett.9b00508

Abstract

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes.

Highlights

The development of optical super-resolution techniques allows researchers to unravel molecular properties of biological systems with far unprecedented detail.[1−4] While recent technical advancements propel the achievable spatial resolution to the true molecular scale of only a few nanometers,[5−7] current implementations are still limited when it comes to imaging many molecular species simultaneously in single cells and beyond
While spectral multiplexing approaches are relatively straightforward to implement, the amount of “plex” is inherently limited by the number of distinguishable spectral labels in the detectable emission spectrum, which is in most instances three or four.[8]
Some efforts to extend spectral multiplexing capabilities include multiparameter and combinatorial detection,[9,10] multispectral acquisition,[11] and spectrally resolved detection.[12,13]. While these approaches increase the number of detectable targets, they are still limited by the spectral properties of fluorescent molecules used to label target structures

Summary

Nano Letters

Exemplary intensity versus time traces from highlighted regions in d representing each of the four unique DNA origami species. (f) Engineering frequency and duration on DNA origami below the diffraction limit. By combining four distinguishable binding frequencies with three spectral colors, we should be able to achieve simultaneous, 124-plex super-resolution imaging within a few minutes acquisition time To demonstrate that this is feasible, we designed and constructed 124 unique DNA origami structures carrying 0, 3, 9, 22, or 44 copies of three orthogonal binding sites each, respectively (Figure 3a and Supplementary Tables 9 and 10). We were able to clearly distinguish four distinct frequency populations in each of the three spectral colors Using these levels, we assigned a unique barcode ID to each of the origami structures in the sample based on color and frequency and were able to render a full 124 pseudocolor super-resolution data set (Figure 3c, see Methods in the Supporting Information for identification).

■ ACKNOWLEDGMENTS

Findings

■ REFERENCES