Mechanical forces play a vital role in biology. Despite their importance, a barrier in their understanding has been made by the challenge to image cellular force with sub-100-nm resolution. Now, scientists led by chemists at Emory University have developed a new technique using tools made of luminescent DNA to visualize the mechanical forces of cells at the molecular level. The technique was demonstrated on human blood platelets.

This work is published in Nature Methods in an article titled, “Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.”

“Normally, an optical microscope cannot produce images that resolve objects smaller than the length of a light wave, which is about 500 nm,” said Khalid Salaita, PhD, professor of chemistry at Emory University and senior author of the study. “We found a way to leverage recent advances in optical imaging along with our molecular DNA sensors to capture forces at 25 nm. That resolution is akin to being on the moon and seeing the ripples caused by raindrops hitting the surface of a lake on the Earth.”

To do this, the researchers turned strands of synthetic DNA into molecular tension probes that contain hidden pockets. The probes are attached to receptors on a cell’s surface. Free-floating pieces of DNA tagged with fluorescence serve as imagers. As the unanchored pieces of DNA whizz about they create streaks of light in microscopy videos.

The synthetic DNA probes (light brown) are anchored to a cell surface (in teal). The free-floating DNA imagers are represented in fluorescent green.
[Pushkar Shinde]
The authors present “tension points accumulation for imaging in nanoscale topography (tPAINT), which integrates molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map pico newton mechanical events with ~25-nm resolution.”

When the cell applies force at a particular receptor site, the attached probes stretch out causing their hidden pockets to open and release tendrils of DNA that are stored inside. The free-floating pieces of DNA are engineered to dock onto these DNA tendrils. When the fluorescent DNA pieces dock, they are briefly demobilized, showing up as still points of light in the microscopy videos.

Hours of microscopy video are taken of the process, then speeded up to show how the points of light change over time, providing the molecular-level view of the mechanical forces of the cell.

The team reported an additional, second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. The authors noted that they “applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts.”

The researchers used a firefly analogy to describe the process.

“Imagine you’re in a field on a moonless night and there is a tree that you can’t see because it’s pitch black out,” said Joshua Brockman, PhD, a former member of the Salaita lab. “For some reason, fireflies really like that tree. As they land on all the branches and along the trunk of the tree, you could slowly build up an image of the outline of the tree. And if you were really patient, you could even detect the branches of the tree waving in the wind by recording how the fireflies change their landing spots over time.”

“It’s extremely challenging to image the forces of a living cell at a high resolution,” said Hanquan Su, PhD, a postdoctoral fellow in the Salaita lab. “A big advantage of our technique is that it doesn’t interfere with the normal behavior or health of a cell.” Another advantage, he added, is that DNA bases of A, G, T, and C, which naturally bind to one another in particular ways, can be engineered within the probe-and-imaging system to control specificity and map multiple forces at one time within a cell.

“Ultimately, we may be able to link various mechanical activities of a cell to specific proteins or to other parts of cellular machinery,” Brockman said. “That may allow us to determine how to alter the cell to change and control its forces.”

Importantly, the tPAINT mechanism also revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

By using the technique to image and map the mechanical forces of platelets, the cells that control blood clotting at the site of a wound, the researchers discovered that platelets have a concentrated core of mechanical tension and a thin rim that continuously contracts. “We couldn’t see this pattern before but now we have a crisp image of it,” Salaita said. “How do these mechanical forces control thrombosis and coagulation? We’d like to study them more to see if they could serve as a way to predict a clotting disorder.”

Just as increasingly high-powered telescopes allow us to discover planets, stars, and the forces of the universe, higher-powered microscopy allows us to make discoveries about our own biology.

“I hope this new technique leads to better ways to visualize not just the activity of single cells in a laboratory dish, but to learn about cell-to-cell interactions in actual physiological conditions,” Su said. “It’s like opening a new door onto a largely unexplored realm—the forces inside of us.”

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