Optical imaging of individual biomolecules in densely packed clusters

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

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA / chemistry*
  • DNA / ultrastructure
  • Microscopy, Fluorescence
  • Molecular Imaging / methods*
  • Nanostructures / chemistry*
  • Nanostructures / ultrastructure
  • Optical Imaging / methods*

Substances

  • DNA