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Nat Nanotechnol. 2016 Sep;11(9):798-807. doi: 10.1038/nnano.2016.95. Epub 2016 Jul 4.

Optical imaging of individual biomolecules in densely packed clusters.

Dai M1,2, Jungmann R1,3, Yin P1,3.

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Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.
Biophysics Program, Harvard University, Boston, Massachusetts 02115, USA.
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.


Recent advances in fluorescence super-resolution microscopy have allowed subcellular features and synthetic nanostructures down to 10-20 nm in size to be imaged. However, the direct optical observation of individual molecular targets (∼5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography)-a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization-on synthetic DNA nanostructures. We examined the effects of a high photon count, high blinking statistics and an appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (root mean squared) over hours. This allowed us to image a densely packed triangular lattice pattern with ∼5 nm point-to-point distance and to analyse the DNA origami structural offset with ångström-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed exchange-PAINT imaging, we further demonstrated an optical nanodisplay with 5 × 5 nm pixel size and three distinct colours with <1 nm cross-channel registration accuracy.

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