Uncurling a single DNA molecule and gluing it down helps sharpen images

14 Jan 2025, 16:00 by American Institute of Physics

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Fluorescence observation of a single DNA molecule migrating in the microchannel and schematic of the stretch and immobilization processes. The DNA molecule was stained with YOYO-1. After one DNA molecule was trapped on the surface treated by APTES, the DNA molecule was stretched by pressure flow and immobilized. Credit: AIP Advances (2025). DOI: 10.1063/5.0223375

Most microscopes can only illuminate objects down to a certain size before tiny features blur together. This blurring is known as the diffraction limit of light. Super-resolution imaging techniques, however, can distinguish between tiny biomolecular features, especially when thermal fluctuations are minimized.

Using advanced imaging techniques and precise microfluidics control to stretch out curly DNA into a straight line, research published in AIP Advances demonstrates techniques for stretching and immobilizing DNA with minimum thermal fluctuation to enable detailed analysis.

"Immobilizing the molecule essentially 'glues' it to a substrate, preventing any movement caused by thermal fluctuations," author Naoki Azuma said. "Super-resolution imaging often requires just seconds or minutes to capture the image. During this time, thermal fluctuations—random vibrations caused by the molecule's thermal energy—result in blurry images and decreased lateral resolution."

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 at Nagoya University 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 specialized chemical that creates chemical bonds between the DNA and a glass substrate to "glue" the DNA molecule in place.

"While it is not yet possible to directly visualize 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 immobilizing DNA molecules for more accurate analysis."

More information: Naoki Azuma et al, Stretching and immobilizing a single DNA molecule on a glass surface using pressure flow in a microchannel for super-resolution imaging, AIP Advances (2025). DOI: 10.1063/5.0223375

Journal information: AIP Advances

Provided by American Institute of Physics