Uncurling and 'gluing down' DNA molecules for sharper imaging

Most microscopes can only illuminate objects down to a certain size before their features blur together. However, super-resolution imaging can distinguish between biomolecular features, especially when thermal fluctuations — random vibrations caused by the molecule’s thermal energy — are minimised.

Now, researchers at Nagoya University have demonstrated techniques for stretching and immobilising DNA with minimum thermal fluctuation to enable detailed analysis. Their work has been published in the journal AIP Advances.

“Super-resolution imaging often requires just seconds or minutes to capture the image. During this time, thermal fluctuations … result in blurry images and decreased lateral resolution,” said study author Naoki Azuma.

“Immobilising the molecule essentially ‘glues’ it to a substrate, preventing any movement caused by thermal fluctuations.”

Researchers have previously tried sticking down one end of a DNA molecule to stretch it out, but found that the thermal fluctuations could still cause movement and blurring.

“Stretching DNA refers to the process of stretching a single DNA molecule, which is originally coiled in a random coil, into a straight line,” Azuma said. “The length and structure of a single DNA molecule, its specific base sequence, and its interactions with proteins must be observed by stretching it for detailed analysis.”

Azuma and his colleagues experimented with ways to uncurl a DNA molecule using pressure applied to liquid flowing in a channel, with the pressure flow providing shear force that uncurled the DNA molecule. They found that controlling the flow velocity of the liquid helps fine-tune the shear force applied and allows precise adjustments of the stretch ratio of the DNA.

Controlling the stretch ratio was a key factor for accurate analysis. In the process, they also used a specialised chemical that creates chemical bonds between the DNA and a glass substrate to keep the DNA molecule in place.

“While it is not yet possible to directly visualise individual base pairs, these methods enable much higher precision in observing molecular-scale structures,” Azuma said. “We aim to refine these methods to achieve higher fidelity in stretching and immobilising DNA molecules for more accurate analysis.”

Image caption: Fluorescence image of DNA molecules with YOYO-1 fluorescent dye in the microchannel after applying the pressure flow. Although many DNA molecules were stretched and immobilised, there were many DNA molecules with a low degree of stretch. Image courtesy of the study authors under CC BY-NC-ND 4.0