How light can reveal the hidden orientation of a single molecule
Matthew Lew’s lab crafts imaging technology to learn molecular details
By looking closely at a single molecule, researchers can learn more about how proteins and cell membranes are organized and how molecules behave in cells. But getting a good image is not as simple as it might sound — the direction the molecule is facing makes a difference.
Alex Nie, who earned a bachelor’s degree in electrical engineering from McKelvey Engineering in 2026 while working in the lab of Matthew D. Lew, associate professor in the Preston M. Green Department of Electrical & Systems Engineering in the McKelvey School of Engineering at Washington University in St. Louis, found that polarized light patterns can improve how precisely microscopes measure the orientation of single molecules. Their research was published Aug. 26, 2026, in Biomedical Optics Express.
From a molecule’s orientation, researchers can learn how molecules are arranged within a larger structure of which they are a part, how they interact with other structures and whether they are moving or wobbling. In other words, the orientation provides researchers with more information about structure, motion and interactions than studying a molecule’s position alone.
The microscope in Lew’s lab is designed to measure the 3D position and 3D orientation of a single molecule using fluorescence. Their goal was to determine the best angle at which to shine light on the molecule; for example, to find the best light polarization that would reveal more about its orientation, aiming to hit the fundamental physical limits of performance of the imaging system, the researchers said.
“Ordinary microscopes only tell you where a molecule is,” said Nie, who has received a Dean’s Select Fellowship from the McKelvey School of Engineering to begin his doctoral studies in Imaging Science at WashU in spring 2027. “With our microscope, we also get color, and that color tells us which way the molecule is pointing. When we know that, we can model those emitters as a dipole.”
Lew said his lab has been determining the best way to image an object for some time but had not explored how illuminating a sample could change the imaging sensitivity.
“If you illuminate the object the right way, then you can dramatically change how accurately you can measure, not only where the molecules are but how they're oriented,” Lew said. “There's an engineering principle called Fisher information for determining how much information is present in the image from the microscope, and once we know how much information there is, we can design a scheme to maximize it.”
The technology is so precise that it can view the organization of peptides in amyloid fibers, which are connected to Alzheimer's disease, Parkinson's disease and type II diabetes, and is able to image the structure of biomolecular condensates.
“We need as much precision as we can get in any particular imaging experiment to resolve what's happening,” Lew said. “Alex's technology is not just a question of improving resolution, but it extends the frontier of what we’re able to see, because we're at that limit where molecules are moving just a little too quickly and are just a little too dim. We don't get the data we need unless we really optimize the scope, and Alex’s work pushes us all forward.”
Nie KA, Qiu Y, and Lew MD. Optimal excitation polarization schemes to boost the precision of single-molecule orientation imaging. Biomedical Optics Express 17, 4854-4869 (2026). https://doi.org/10.1364/BOE.607950
This research was supported by the National Institutes of Health’s National Institute of General Medical Sciences (R35GM124858).
The excitation modulation optimization methodology mentioned in this work was invented by Nie, Qui and Lew. Washington University in St. Louis has filed a provisional patent application covering the technology.
