Existing super-resolution microscopy systems are already good at observing and tracking individual molecules within intact cells. However, they need to get better at detecting the interactions between molecules. Especially important are the interactions between signaling molecules. These interactions occur at a scale at least four times smaller than that resolved by existing single-molecule microscopes.

Detecting these interactions often amounts to determining, with great precision, the spatial arrangements of molecules—or, more simply, measuring the distances between interacting molecules. Localizing individual molecules in the cellular context promises to revolutionize biology, and has been pursued through techniques such as (direct) stochastic optical reconstruction microscopy (STORM) and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT).

Both techniques have been refined to allow the collection of sequential images, which can be analyzed together to yield localization information, albeit at the cost of long image-acquisition times, which means that drifting molecules may be hard to pin down. To help overcome this problem, scientists at the University of New South Wales (UNSW), led by Scientia Professor Katharina Gaus, PhD, have developed a new approach to single-molecule localization microscopy (SMLM). They call it Feedback SMLM.

The new approach is described in a paper (“Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells”) that appeared in Science Advances. According to this article, Feedback SMLM performs real-time drift correction in three dimensions.

“Our actively stabilized microscope achieves a stabilization of <1 nm and localization precision of ~1 nm,” the article’s authors wrote. “To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T-cell receptors and phosphatases (CD45) in active and resting T cells.”

Essentially, the scientists built autonomous feedback loops inside a single-molecule microscope that detects and realigns the optical path and stage.

“It doesn’t matter what you do to this microscope, it basically finds its way back with precision under a nanometer,” said Gaus, who is also head of UNSW Medicine’s EMBL Australia Node in Single Molecule Science. “It’s a smart microscope. It does all the things that an operator or a service engineer needs to do, and it does that 12 times per second.”

Because Feedback SMLM does not require filtering, merging, averaging, or other post-acquisition corrections, the molecular emission “landscape” created by successive rebinding or photoactivation/switching events reflects their true spatial and structural arrangement.

“The localization precision of single-molecule microscopes is [normally] around 20–30 nanometers,” noted Gaus. “[That’s because] the microscope actually moves while we’re detecting that signal. This leads to an uncertainty. With the existing super-resolution instruments, we can’t tell whether or not one protein is bound to another protein because the distance between them is shorter than the uncertainty of their positions.”

With the design and methods outlined in the paper, the feedback system designed by the UNSW team is compatible with existing microscopes and affords maximum flexibility for sample preparation.

“It’s a really simple and elegant solution to a major imaging problem,” asserted Gaus. “We just built a microscope within a microscope, and all it does is align the main microscope. That the solution we found is simple and practical is a real strength as it would allow easy cloning of the system, and rapid uptake of the new technology.”

To demonstrate the utility of their ultraprecise feedback single-molecule microscope, the researchers used it to perform direct distance measurements between signaling proteins in T cells. A popular hypothesis in cellular immunology is that these immune cells remain in a resting state when the T-cell receptor is next to another molecule that acts as a brake.

Their high precision microscope was able to show that these two signaling molecules are in fact further separated from each other in activated T cells, releasing the brake and switching on T-cell receptor signaling.

“Conventional microscopy techniques would not be able to accurately measure such a small change as the distance between these signaling molecules in resting T cells and in activated T cells only differed by 4–7 nanometers,” noted Gaus. “This also shows how sensitive these signaling machineries are to spatial segregation. To identify regulatory processes like these, we need to perform precise distance measurements, and that is what this microscope enables. These results illustrate the potential of this technology for discoveries that could not be made by any other means.”

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