WashU Expert: Optical microscopes take a big leap forward

What if piggybacking on a molecule with a fluorescent marker could tell us not just where the molecule is but how it’s oriented?

The technique could find a whole host of uses — from dynamically tracking cell behavior and understanding the mechanics of Alzheimer’s disease, to studying how polymer layers move in smart materials.

Powering traditional workhorses

Matthew Lew, associate professor of electrical & systems engineering in the Preston M. Green Department of Electrical & Systems Engineering in the McKelvey School of Engineering at Washington University in St. Louis, has pioneered such a technique, known as single-molecule orientation and localization microscopy (SMOLM). A paper in Nature Photonics is the culmination of years of his work into the methodology, which vastly improves the utility of traditional optical microscopes. 

The vision that drives Lew’s lab is to make optical microscopes more potent tools in understanding biological and chemical processes. One path to that vision is to attach a fluorophore to a molecule and study its emission patterns using a polarizing microscope, which gives us information about the attached molecule’s orientation. (Each emission pattern is a signal for a particular molecular orientation so studying them helps deduce orientation).

Lew’s method not only gives information about a molecule’s orientation, but it can also track it dynamically over time. Contrast this ability against traditional ultrahigh-resolution approaches like cryo-electron microscopy, that must literally freeze an object to produce atomic-resolution images. By enabling researchers to observe both molecular orientation and how it changes over time, Lew’s work dramatically expands the limits of optical microscopy.

Why study molecule orientation in the first place?

To make the materials it needs, cells must manipulate molecules and determine not just where to place them — but how. To build proteins for example, long sequences of amino acids have to fold (or not) in specific ways. Building a microscope to measure molecular orientations could be the key to studying how protein conformations impact health and disease.

“There are questions related to molecular orientation that we’ve never been able to answer before, because we just never had the technology to look at how something is pointed in space. Most microscopes are only concerned with producing images of where molecules are,” Lew said.

One of those questions relates to the quandary of the amyloid-beta molecule. While the molecule is a signature of Alzheimer’s disease, simply eliminating it does not prevent disease onset. Instead, the way the amyloid-beta molecules aggregate, form fibrous extensions and grow in layers could be significant in understanding the mechanics of the disease.

Another big question: How rotational freedom of the receptor on an immune cell enables it to search for and find its target. It gained prominence during the COVID-19 pandemic when researchers found that the virus needed a special orientation fit with the proper cellular receptor to activate a binding mechanism and do its work. 

How to make the fluorophore and molecule dance together

To be able to measure a molecule’s orientation and track it over time, the fluorophore needs to steadfastly attach to it, head and tail. After all, wobbling from the fluorophore might be misconstrued as coming from the molecule being studied. 

Chemistry comes to the rescue. A covalent bond attaches the fluorophore to the molecule. Unfortunately, chemistry is not a solution for every molecule of interest.

To get around the labeling problem, Lew and team exploit another characteristic of fluorophores: They are excellent sensors.

The fluorophores used to attach to amyloid aggregates, for example, can sense the aggregates’ sticky nature. Once they see an amyloid fiber, they stick to it and light up, so researchers can readily measure its position and orientation. “The use of a fluorescent molecule as a sensor, where it will only turn on near the target we want to measure, has allowed us to get around labeling difficulties,” Lew said.

A promising range of applications

In addition to using this microscopy technique in biomedicine, Lew is wielding it to study engineered DNA origami that can scaffold and arrange groups of fluorescent molecules. If arranged properly, these molecules could become entangled—a unique phenomenon in quantum mechanics where the behavior of individuals cannot be described independently from the rest of the group — that can be useful in new kinds of quantum sensing and information storage.

Lew says he is excited about the advances his team has systematically delivered over the years.

“About 10 years ago, this technology was in its infancy. We’ve not only built the first versions of the optical hardware and imaging algorithms but also pioneered the first applications in a whole bunch of different areas,” Lew said.

It’s not just AI that’s going to push our knowledge of biomedicine, he pointed out.

“Here we’re building fundamental technologies to see and measure properties of molecules that play surprising and intricate roles,” he said. “We’re excited to see the different applications not just in biology but in a range of other fields.”

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Brasselet S, Lew, MD. Single-molecule orientation and localization microscopy. Nature Photonics, Sept. 1, 2025. DOI: https://doi.org/10.1038/s41566-025-01724-y

This work was funded by Agence Nationale de la Recherche (ANR-20-CE42-0003, ANR-21-CE24-0014, France 2030 Investment Plan IDEC ANR-21-ESRE-0002, Investissements d’Avenir CENTURI ANR-16-CONV-0001, France BioImaging National Infrastructure ANR-10-INBS-04 to S.B.) and the National Institutes of Health (R35GM124858 to M.D.L.).

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