Self-aligning microscope detects molecular interactions

The 2014 Nobel Prize in Chemistry was awarded for the development of super-resolution fluorescence microscopy technology that afforded microscopists the first molecular view inside cells — a capability that has provided new molecular perspectives on complex biological systems and processes. Now the limit of detection of single-molecule microscopes has been smashed again, thanks to the development of an ultraprecise microscope that is capable of detecting interactions between individual molecules within intact cells.

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. As explained by Scientia Professor Katharina Gaus, Head of UNSW Medicine’s EMBL Australia node in Single Molecule Science, “The reason why the localisation precision of single-molecule microscopes is around 20–30 nm 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.”

To circumvent this problem, Prof Gaus and her team built autonomous feedback loops inside a single-molecule microscope that detects and realigns the optical path and stage. She explained, “It doesn’t matter what you do to this microscope, it basically finds its way back with precision under a nanometre. 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.”

As described in the journal Science Advances, the feedback system designed by the UNSW team is compatible with existing microscopes and affords maximum flexibility for sample preparation, resulting in what Prof Gaus described as “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.”

To demonstrate the utility of their ultraprecise 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. The 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 nm,” Prof Gaus said.

“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.”

Image caption: A T cell with precise localisation of T cell receptors (pink) and CD45 phosphatase (green). Image credit: Single Molecule Science.

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