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New super-resolution microscopy method approaches the atomic scale

New super-resolution microscopy method approaches the atomic scale

Location AFM and X-ray structure. Credit: Weill Cornell Medicine, University of Leeds & Washington University

Researchers at Weill Cornell Medicine have developed a computational technique that greatly increases the resolution of atomic force microscopy, a specialized microscope shape that “marks”

; the atoms on a surface. The method reveals details at the atomic level about proteins and other biological structures under normal physiological conditions, which opens a new window on cell biology, virology and other microscopic processes.

In a study published on June 16 in Nature, investigators describe the new technique, which is based on a strategy used to improve resolution in light microscopy.

To examine proteins and other high-resolution biomolecules, researchers have long relied on two techniques: X-ray crystallography and cryoelectron microscopy. While both methods can determine molecular structures down to the dissolution of individual atoms, they do so on molecules that are either scaffolded in crystals or frozen at ultra-cold temperature, possibly altering them from their normal physiological forms. Atomic force microscopy (AFM) can analyze biological molecules under normal physiological conditions, but the resulting images have been blurred and low resolutions.

“Atomic force microscopy can easily solve atoms in physics, on solid surfaces of silicates and on semiconductors, so that means the machine in principle has the precision to do so,” said senior author Dr. Simon Scheuring, Professor of Physiology and Biophysics in Anesthesiology at Weill Cornell Medicine. “The technique is a bit like if you were to take a pen and scan over the Rocky Mountains so you get a topographic map of the object. In reality, our pen is a needle that is sharp down to a few atoms, and objects are single-protein molecules.”

However, biological molecules have many small parts that wiggle and blur their AFM images. To solve this problem, Dr. Scheuring and his colleagues a concept from light microscopy called super-resolution microscopy. “Theoretically, it was not possible by optical microscopy to solve two fluorescent molecules that were closer to each other than half the wavelength of light,” he said. However, by stimulating the adjacent molecules to fluoresce at different times, microscopists can analyze the scattering of each molecule and locate their locations with high precision.

Instead of stimulating fluorescence, Dr. Scheuring’s team found that the natural fluctuations in biological molecules recorded during AFM scans provide similar ranges of position data. First author Dr. George Heath, who was a postdoctoral fellow at Weill Cornell Medicine at the time of the study and now a faculty member at the University of Leeds, engaged in experiments and computational simulations to better understand the AFM imaging process and extract the maximum amount of information from the atomic interactions between peaks. and try.

Using a method such as super-resolution analysis, they were able to extract images with much higher resolution of the moving molecules. Continuing the topographic analogy, Dr. explained. Scheuring that “if the rocks (ie atoms) wiggle a little up and down, you can detect this, then one, and then you average all detections over time, and you receive high-resolution information.”

Because previous AFM surveys have routinely collected the necessary data, the new technique can be applied retroactively to the blurred images the field has generated over decades. As an example, the new paper includes an analysis of an AFM scan of an aquaporin membrane protein originally acquired under Dr. Scheuring’s doctoral dissertation. The gene analysis generated a much sharper image that closely matches X-ray crystallography structures of the molecule. “You’re basically getting quasi-atomic solution on these surfaces now,” said Dr. Splitting. To demonstrate the power of the method, the authors provide new high-resolution data on annexin, a protein involved in cell membrane repair, and on a proton chloride antiporter, where they also report structural changes related to its functional.

In addition to allowing researchers to study biological molecules under physiologically relevant conditions, the new method has other benefits. For example, X-ray crystallography and cryoelectron microscopy rely on averages of data from a large number of molecules, but AFM can generate images of single molecules. “Instead of having observations of hundreds of molecules, we observe a molecule a hundred times and compute a high-resolution map,” said Dr. Splitting.

Imaging of individual molecules as they perform their functions could open up entirely new types of assays. “Let’s say you have one [viral] spike protein that is in one conformation and then it is activated and goes into another conformation, “said Dr. Scheuring. You would in principle be able to calculate a high resolution map from the same molecule as it is transferred from one conformation to the next, not from thousands of molecules in one or the other conformation. “Such high-resolution single-molecule data could provide more detailed information and avoid the potentially misleading results that can occur when calculating averaging data from many molecules. In addition, the map can reveal new strategies for accurately redirecting or interrupting such processes.

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More information:
George R. Heath et al., Localization atomic force microscopy, Nature (2021). DOI: 10.1038 / s41586-021-03551-x

Provided by Weill Cornell Medical College

Citation: New super-resolution microscopy method approaches the atomic scale (2021, June 16) retrieved June 16, 2021 from https://phys.org/news/2021-06-super-resolution-microscopy-method-approaches-atomic.html

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