Dai, MingjieJungmann, RalfYin, Peng2017-02-182016Dai, Mingjie, Ralf Jungmann, and Peng Yin. 2016. “Optical visualisation of individual biomolecules in densely packed clusters.” Nature nanotechnology 11 (9): 798-807. doi:10.1038/nnano.2016.95. http://dx.doi.org/10.1038/nnano.2016.95.1748-3387http://nrs.harvard.edu/urn-3:HUL.InstRepos:30370915Recent advances in fluorescence super-resolution microscopy have allowed sub-cellular features and synthetic nanostructures down to ~15 nm in size to be imaged. However, 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 hybridisation - on synthetic DNA nanostructures. We examined the effects of high photon count, high blinking statistics, and appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. This allowed us to image a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analyse DNA origami structural offset with angstrom-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed Exchange-PAINT imaging, we further demonstrated an optical nano-display with 5×5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy. This method opens up possibilities for direct and quantitative optical observation of individual biomolecular features in crowded environments.en-USJournal Article2017-02-1810.1038/nnano.2016.95