Super optical microscope and DNA-PAINT used to distinguish features 5 nanometers apart

A team at Harvard’s Wyss Institute for Biologically Inspired Engineering led by Core Faculty member Peng Yin, Ph.D., has, for the first time, been able to tell apart features distanced only 5 nanometers from each other in a densely packed, single molecular structure and to achieve the so far highest resolution in optical microscopy. Reported on July 4 in a study in Nature Nanotechnology, the technology, also called “discrete molecular imaging” (DMI), enhances the team’s DNA nanotechnology-powered super-resolution microscopy platform with an integrated set of new imaging methods.

Discrete molecular imaging (DMI). Top: four technical requirements for achieving DMI. Bottom: DNA-PAINT images of 5 nm grid, “Wyss!” pattern and three-colour 5 nm grid structures, all with ∼5 nm pixel size. The 5 nm grid: for each representative single-molecule image, the fluorescent super-resolution image (left) and the automatically fitted image (right) are shown. The “Wyss!” pattern: the single-particle class average (left) and an automatically fitted single-molecule image with overlaid design pattern (green circles) are shown. The three-colour 5 nm grid: for each representative single-molecule image, the automatically fitted images are shown for the three imaging channels separately (left three columns) and for the composite image (rightmost column). It is important to note that no prior knowledge of the sample structures (the 5 nm grid, “Wyss!” pattern, or three-colour 5 nm grid) was used to produce the above results.

Nature Nanotechnology -Optical imaging of individual biomolecules in densely packed clusters

Last year, the opportunity to enable researchers with inexpensive super-resolution microscopy using DNA-PAINT-based technologies led the Wyss Institute to launch its spin-off Ultivue Inc.

“The ultra-high resolution of DMI advances the DNA-PAINT platform one step further towards the vision of providing the ultimate view of biology. With this new power of resolution and the ability to focus on individual molecular features, DMI complements current structural biology methods like X-ray crystallography and cryo-electron microscopy. It opens up a way for researchers to study molecular conformations and heterogeneities in single multi-component complexes, and provides an easy, fast and multiplexed method for the structural analysis of many samples in parallel” said Peng Yin, who is also Professor of Systems Biology at Harvard Medical School.

DNA-PAINT super-resolution experiment setup and workflow

DNA-PAINT technologies, developed by Yin and his team are based on the transient binding of two complementary short DNA strands, one being attached to the molecular target that the researchers aim to visualize and the other attached to a fluorescent dye. Repeated cycles of binding and unbinding create a very defined blinking behavior of the dye at the target site, which is highly programmable by the choice of DNA strands and has now been further exploited by the team’s current work to achieve ultra-high resolution imaging.

“By further harnessing key aspects underlying the blinking conditions in our DNA-PAINT-based technologies and developing a novel method that compensates for tiny but extremely disruptive movements of the microscope stage that carries the samples, we managed to additionally boost the potential beyond what has been possible so far in super-resolution microscopy,” said Mingjie Dai, who is the study’s first author and a Graduate Student working with Yin.


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 less than 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 48 pages of supplemental material

SOURCES -Nature Nanotechnology, Eurekalert, Harvard