Self-Aligning Microscope with Super-Resolution

Researchers at the University of New South Wales have achieved unprecedented resolution capabilities in single-molecule micro­scopy to detect inter­actions between indi­vidual molecules within intact cells. After the development of super-resolution fluores­cence micro­scopy tech­nology the limit of detection of single-molecule microscopes has been smashed again. While indi­vidual molecules could be observed and tracked with super-reso­lution micro­scopy already, inter­actions between these molecules occur at a scale at least four times smaller than that resolved by existing single-molecule micro­scopes.

A T cell with precise localisation of T cell receptors (pink) and CD45 phosphatase. (Source: Single Molecule Science, UNSW)

“The reason why the loca­lisation precision of single-molecule microscopes is around 20-30 nanometers normally is 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,” says Katharina Gaus, research team leader.

To circumvent this problem, the team built auto­nomous feedback loops inside a single-molecule micro­scope that detects and re-aligns 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. 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,” says Gaus.

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. 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,” says Gaus.

To demonstrate the utility of their ultra-precise feedback single-molecule micro­scope, the researchers used it to perform direct distance measure­ments between signalling 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 signalling molecules are in fact further separated from each other in acti­vated T cells, releasing the brake and switching on T cell receptor signalling.

“Conven­tional micro­scopy techniques would not be able to accu­rately measure such a small change as the distance between these signalling molecules in resting T cells and in activated T cells only differed by 4-7 nano­meters,” says Gaus. “This also shows how sensitive these signalling machineries are to spatial segre­gation. In order 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.” (Source: UNSW)

Reference: S. Coelho et al.: Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells, Sci. Adv. 6, eaay8271 (2020); DOI: 10.1126/sciadv.aay8271

Link: Single Molecule Science, University of New South Wales, Sydney, Australia

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