Australian scientists have reported achieving new levels of resolution in single-molecule microscopy, allowing them to detect interactions between individual molecules within intact cells.
They say the capabilities of their self-aligning microscope exceed those of the technology which won the 2014 Nobel prize in chemistry, and which afforded microscopists the first molecular view inside cells.
The breakthrough by medical researchers from UNSW is described in a paper in the journal Science Advances.
Research leader Katharina Gaus says while it is possible to follow individual molecules with existing instruments, they struggle to track interactions between molecules.
“The reason why the localisation precision of single-molecule microscopes is around 20-30 nanometres normally is because the microscope actually moves while we’re detecting that signal,” she says.
“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, 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 nanometre,” Gaus says. “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.”
To demonstrate the microscope’s utility, the researchers performed 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 microscope was able to show that the 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 four to seven nanometres,” says Gaus.