Expansion microscopy tool kit corrects distortion of blown-up cells

Biologists depend on microscopes to see what their cell samples are up to, but the smallest organelles are hard to resolve with conventional methods. To get a better look at very small things, biologists can make their samples bigger by swelling them with a hydrogel—a technique called expansion microscopy (ExM).

But organelles rarely swell evenly; for example, mitochondria don’t expand as much as the surrounding cytosol. That distortion lowers the fidelity of ExM images. A new tool kit could help overcome this limitation by using nanosize rulers and digital image analysis to correct for distortions and reconstruct the sample in a 3D image (ACS Nano 2026, DOI: 10.1021/acsnano.6c00277).

“It’s one thing to make [ExM] work but another thing to make it a really quantitative and reliable method,” says Izzy Jayasinghe, a molecular biologist at the University of New South Wales who coauthored the study. ExM could overcome the diffraction limits of a microscope, but determining the extent to which a specimen expands—also known as its expansion factor—can be challenging, Jayasinghe adds.

The gels typically used to swell ExM specimens often can’t penetrate into confined structures such as endosomes, which are only 100–300 nm in diameter, Jayasinghe says. This limitation often leads to underexpansion of these compartments.

To visualize minuscule organelles without losing track of these distortions, Jayasinghe, Tayla Shakespeare of the University of Lisbon, and coworkers genetically modified the cells they were analyzing to build nanosize rulers. Specifically, the researchers introduced a gene that allowed the cells to build soccer ball–shaped protein nanocages. These tiny polyhedral protein balls expand to a similar degree as the small organelles around them, so by measuring the nanocages in their expanded sample, the researchers could tell how much each part of their cells had swelled.

“For the ability to calibrate your expansion factor in gel, this is the best way to do it at the moment,” says Benjamin Liffner, a cell biologist at Adelaide University who uses ExM to study parasites but wasn’t involved in the study. The technique offers a fine-grained analysis without compromising usability and ease, he adds.

During the sample preparation, the researchers let the gel expand about 1,000-fold. This translates to a 10-fold magnification of the samples in 3D.

Using the tool kit, the team then achieved resolution high enough to visualize endosomes shuttling cargo into the cell’s cytoplasm. The nanocage measurements, along with a distortion analysis and a digital tracing software, helped the scientists reconstruct the endosomes’ original 3D form.

Using images of cells taken 15 min apart, the team was able to model how signaling proteins cluster on the endosomes’ surface. The researchers even quantified how some of these proteins move into the interior of the organelle as the endosome navigates toward the center of the cell—a process that “hadn’t been really characterized before” because of the low resolution, Jayasinghe says.

The combination of the gel expansion and an Airyscan confocal microscope, which has a resolution up to 1.7 times as high as a conventional microscope, puts ExM “well into the realm of other types of super-resolution microscopy,” Jayasinghe says. “The improvement in resolution is actually not just in the x and y [planes], but also in the z.” This feature makes ExM a superior method for imaging small compartments in 3D, she says.

Jayasinghe thinks the tool kit can be adapted for other applications, including nonmammalian cells and tissues, as long as the samples can be genetically modified. “It is a very versatile technique,” she says. The team is working toward finding more applications for its ExM tool kit and using it to look at whole organisms.