New computational technology greatly improves the resolution of atomic force microscopes

Scientists at Weill Cornell Medicine have developed computational techniques that significantly improve the resolution of atomic force microscopes. This is a special type of microscope that “feels” the atoms on the surface.

This method reveals atomic-level details about proteins and other biological structures under normal physiological conditions and opens new windows on cell biology, virology, and other microscopic processes.

In one study, on June 16th Nature, Researchers describe new technologies based on strategies used to improve the resolution of light microscopes.

To study proteins and other biomolecules at high resolution, researchers have long relied on two techniques: X-ray crystallography and cryo-electron microscopy. Both methods allow the molecular structure to be determined down to the resolution of individual atoms, but may be determined by scaffolding the crystal or by molecules that freeze at ultra-low temperatures, changing from the normal physiological shape.

Atomic force microscopy (AFM) can analyze biomolecules under normal physiological conditions, but the resulting image is blurry and has low resolution.

Atomic force microscopy, in principle, means that machines have the precision to do that because they can easily decompose atoms on physics, solid surfaces of silicates, and semiconductors. “

Dr. Simon Scheuring, Principal Investigator and Professor, Physiology and Biophysics, Anesthesiology, Weil Cornell Medicine

“This technique is a bit like scanning the Rocky Mountains with a pen, so you can get a topographic map of an object. In fact, our pen is a sharp needle up to a few atoms, and the object is It’s a single protein molecule. “

However, biomolecules have many small areas where the AFM image is blurred and shaken. To address that problem, Dr. Schering and his colleagues adopted the concept of light microscopy called super-resolution microscopy.

“Theoretically, an optical microscope couldn’t break down two fluorescent molecules that are closer than half the wavelength of light,” he said. However, by stimulating adjacent molecules to fluoresce at different times, the microscope can analyze the spread of each molecule and locate them with high accuracy.

Instead of stimulating fluorescence, Dr. Scheuring’s team noted that the natural variation of biomolecules recorded during the AFM scan resulted in a similar spread of position data.

Dr. George Heath, a postdoctoral fellow at Weill Cornell Medicine at the time of the study and now a faculty member at the University of Leeds, led the author, engaged in a cycle of experimentation and computational simulation to gain a deeper understanding of the AFM imaging process. Extract maximum information in detail from the interatomic interactions between the chip and the sample.

Using methods such as super-resolution analysis, they were able to extract much higher resolution images of moving molecules. Continuing the terrain analogy, Dr. Scheuring said, “If a rock (that is, an atom) sways a little up and down, it can be detected and then detected. Then, averaging all detections over time, Receive a high value. Resolution information. “

Previous AFM studies gathered the necessary data on a regular basis, so the new technique can be applied retroactively to blurry images that the field has generated over decades. As an example, the new paper includes an analysis of AFM scans of aquaporin membrane proteins, first obtained in Dr. Schering’s dissertation.

The reanalysis produced a much clearer image that closely matched the X-ray crystal structure of the molecule. “Basically, we get quasi-atomic resolution on these surfaces,” says Dr. Scheuring. To demonstrate the power of this method, the authors provide new high-resolution data on annexin, a protein involved in cell membrane repair, and a proton chloride antiporter that also reports structural changes related to its function.

In addition to allowing researchers to study biomolecules under physiologically appropriate conditions, the new method has other advantages. For example, X-ray crystallography and cryo-electron microscopy rely on averaging data from many molecules, while AFM can generate single-molecule images. “Instead of observing hundreds of molecules, we observe one molecule 100 times and calculate a high-resolution map,” says Dr. Scheuring.

Imaging individual molecules as they perform their functions allows for a whole new type of analysis. “Let’s say you have [viral] Peplomers in one conformation are activated into another conformation, “says Dr. Scheuring.

“As a general rule, when moving from one conformation to the next, you can calculate a high-resolution map from the same molecule, not from thousands of molecules in any conformation.” Such a high resolution. Single molecule data provides more detailed information and avoids the misleading consequences that can occur when averaging data from many molecules. In addition, the map may reveal new strategies for accurately redirecting or interrupting such processes.