Nobel Prize-Winning Techniques Help Resolve Imaging Challenge

Using a technique that was recently awarded the 2022 Nobel Prize in chemistry, researchers at Cornell University used expansion microscopy to study lipids, the water-repellent, dynamic components that comprise the walls of cells and organelles. The microscopy technique developed at Cornell, called Lipid Expansion Microscopy (LExM), will enable closer study of biological membranes, which are the site of critical cell signaling and nutrient exchange.

These processes, if disrupted, can lead to disease, according to Cornell researcher Brittany White-Mathieu.

“Being able to image membrane interactions or just how membranes change when they are introduced to certain stimuli can shed light on how cells respond to external things that can be good or bad for our bodies,” White-Mathieu said.

Expansion microscopy, a technique that expands cell components to make them more visible, allows investigators to expand small structures using a polymer system. The investigator uses chemicals to create a polymer containing charged components, called side chains, and introduces the polymer network into the cellular or tissue sample. When the sample is placed in water, the side chains absorb the water and expand through osmosis to make the sample bigger.

Lipids are hydrophobic, or water-repelling, which makes them an effective protective barrier, though a challenge to image with expansion microscopy. According to Cornell professor Jeremy Baskin, the tagging methods used for this type of microscopy don’t work for lipids.

The dynamic nature of membrane formations and processes, sometimes necessary to move the cell’s contents from one side of the membrane to the other, also make the protective lipid membranes difficult to image.

“The problem we’re trying to solve with this method is being able to visualize when different parts of the cell have to nestle up close to one another,” Baskin said.

Because cellular membranes are only a couple of nanometers in size, superresolution imaging techniques are needed to view the membranes in detail.

This image, featured in a 2008 paper in the journal Science that was co-authored by Jeremy Baskin, shows a developing zebra fish larva in which the sugars on the surface of individual cells are fluorescently tagged with copper-free click chemistry. Courtesy of Cornell Chronicle.

The researchers applied the Nobel Prize-winning techniques of click and bio-orthogonal chemistry to expansion microscopy and lipids. Click chemistry, developed by Morten Meldal and K. Barry Sharpless, enables molecules to snap together quickly and efficiently. Bio-orthogonal chemistry expands on click chemistry by applying click reactions to living organisms. Baskin was a doctoral student in the lab of the developer of bio-orthogonal chemistry, Carolyn R. Bertozzi, in the mid-2000s.

The Cornell team developed metabolic labels with clickable labels that produced a targeted chemical reaction, causing two molecules to snap together like two pieces of a jigsaw puzzle.

“Our goal was to develop chemical reactions for tagging molecules inside of living cells,” Baskin said. “They sort of need to click together like the parts of a seatbelt, or like two pieces of a jigsaw puzzle, where the only two pieces that are going to go next to each other are the right two, in a sea of a million other pieces.”

Once the clickable tags were introduced, the cell samples accepted the molecules and incorporated them into lipids. The researchers then deployed a fluorescent reagent that they designed that reacts with the clickable handles on lipids. The reagent contains a polymerizable unit that incorporates into the polymer network during the expansion procedure. When the sample is expanded, the lipid is attached directly to the polymer network through the fluorescent compound.

The researchers used the LExM technique to visualize organelle membranes with precision.

The technique, which is compatible with standard confocal microscopes, could be used for superresolution imaging of phospholipids and cellular membranes in various physiological contexts. The researchers said that it could potentially be used to study genetic diseases associated with perturbed lipid metabolism, an area where further study could help inform the development of future therapies.

Serendipitously, the research was published the same week as the Nobel Prize for click chemistry was awarded.

“It’s one of hundreds or thousands of illustrations of the power of click chemistry,” Baskin said.

The research was published in the Journal of the American Chemical Society (www.doi.org/10.1021/jacs.2c03743).