Pushing the limits of super-resolution microscopy: self-aligning microscopy
Ultra-precision microscopy that goes beyond the limitations of the Nobel Prize-winning super-resolution microscope will allow scientists to directly measure the distances between individual molecules.
Medical researchers at the University of New South Wales have achieved unprecedented resolution in single-molecule microscopy to detect interactions between individual molecules within intact cells.
The 2014 Nobel Prize in Chemistry was awarded for the development of super-resolution fluorescence microscopy technology, which provided microscopists with the first molecular view of the inside of a cell, a feature that provides new molecular views of complex biological systems and processes.
Now, the detection limits of single-molecule microscopy have been pushed once again, and details have been published in the latest issue of Science Advances.
It has been possible to observe and track individual molecules with ultrahigh-resolution microscopes, but the interactions between these molecules occur on a scale that is at least four times smaller than that resolved by existing single-molecule microscopes.
"The reason single-molecule microscopes typically have localization accuracies around 20 to 30 nanometers is usually because the microscope actually moves when detecting signals. This leads to uncertainty. Using existing super-resolution instruments, we can fail to determine whether one protein is bound to another because the distance between them is shorter than the uncertainty in their positions."
To solve this problem, the team built an automated feedback loop inside the single-molecule microscope that detects and realigns the optical path and mirror stage.
"It doesn't matter what you do with this microscope, it basically finds the return path with nanometer precision. It's a smart microscope. It can do everything an operator or service engineer needs to do and it can do that 12 times a second." Prof. Goss said.
Measuring the distance between proteins
With the design and methodology outlined in this paper, the UNSW team has designed a feedback system that is compatible with existing microscopes and provides maximum flexibility for sample preparation.
"This is a very simple and elegant solution to a major imaging problem. We just built a microscope inside a microscope and all we did was align the main microscope. The simplicity and practicality of the solution we found is its real strength. It's easy to clone the system and adopt new technologies quickly." Prof. Goss said.
To demonstrate the utility of its ultra-precise feedback single-molecule microscope, the researchers used it to make direct distance measurements between signaling proteins in T cells. A common assumption in cellular immunology is that these immune cells remain quiescent when the T-cell receptor is close to another molecule that acts as a brake.
Their high-precision microscopy was able to show that the two signaling molecules were actually further separated from each other in activated T cells, releasing the brake and turning on T cell receptor signaling.
Prof. Goss said, "Conventional microscopy techniques would not be able to accurately measure such a small change because the distance between these signaling molecules in quiescent and activated T cells differs by only 4-7 nanometers."
"This also shows how sensitive these signaling mechanisms are to spatial isolation. To identify such regulatory processes, we need to perform precise distance measurements, which is what this microscope enables. These results illustrate that the technology is in discovery and cannot be manufactured in any other way."
