One of the earliest iterations of the microscope was built in the 1600s by the microscopy pioneer Antonie van Leeuwenhoek. His work not only developed the field of microscopy but opened up the study of microbes upon his observation of “wee animalcules”—his description of unicellular organisms. More advanced microscopes have been developed since Leeuwenhoek’s time, but some researchers are innovating new ways to use ordinary light microscopes to delve deeper into the biological world. For example, MIT engineers have devised a technique for imaging biological samples with an accuracy of 10 nm, a scale that should enable them to image viruses and potentially even single biomolecules.

The new technique builds on expansion microscopy (ExM), an approach first developed in 2015, that physically magnifies biological specimens to enable nanoscale-resolution imaging using conventional microscopes. Current ExM methods involve embedding biological samples in a hydrogel and then expanding them before imaging them with a microscope.

This work is published in Nature Nanotechnology in the paper, “A highly homogeneous polymer composed of tetrahedron-like monomers for high-isotropy expansion microscopy.”

In expansion microscopy, researchers physically enlarge their samples about fourfold in linear dimension before imaging them, allowing them to generate high-resolution images without expensive equipment.

“Hundreds of groups are doing expansion microscopy. There’s clearly pent-up demand for an easy, inexpensive method of nanoimaging,” said Edward Boyden, PhD, professor of biological engineering and brain and cognitive sciences at MIT, and a member of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research. “Now the question is, how good can we get? Can we get down to single-molecule accuracy? Because in the end, you want to reach a resolution that gets down to the fundamental building blocks of life.”

In this new version of the technique, researchers have developed a new type of hydrogel that maintains a more uniform configuration, allowing for greater accuracy in imaging structures. They show that ExM is possible using hydrogels that have “a more homogeneous network structure, assembled via non-radical terminal linking of tetrahedral monomers.”

In a 2017 paper, Boyden’s lab demonstrated resolution of around 20 nm, using a process in which samples were expanded twice before imaging. This approach, as well as the earlier versions of expansion microscopy, relies on an absorbent polymer made from sodium polyacrylate, assembled using a method called free radical synthesis. These gels swell when exposed to water; however, one limitation of these gels is that they are not completely uniform in structure or density. This irregularity leads to small distortions in the shape of the sample when it’s expanded, limiting the accuracy that can be achieved.

To overcome this, the researchers developed a new gel called tetra-gel, which forms a more predictable structure. By combining tetrahedral PEG molecules with tetrahedral sodium polyacrylates, the researchers were able to create a lattice-like structure that is much more uniform than the free-radical synthesized sodium polyacrylate hydrogels they previously used.

The researchers demonstrated the accuracy of this approach by using it to expand particles of herpes simplex virus type 1 (HSV-1), which have a distinctive spherical shape. After expanding the virus particles, the researchers compared the shapes to the shapes obtained by electron microscopy and found that the distortion was lower than that seen with previous versions of expansion microscopy, allowing them to achieve an accuracy of about 10 nm.

“We can look at how the arrangements of these proteins change as they are expanded and evaluate how close they are to the spherical shape. That’s how we validated it and determined how faithfully we can preserve the nanostructure of the shapes and the relative spatial arrangements of these molecules,” Ruixuan Gao, PhD, a post-doc in the Boyden lab, and first author on the paper, said.

The researchers also used their new hydrogel to expand cells, including human kidney cells and mouse brain cells. They are now working on ways to improve the accuracy to the point where they can image individual molecules within such cells. One limitation on this degree of accuracy is the size of the antibodies used to label molecules in the cell, which are about 10 to 20 nm long. To image individual molecules, the researchers would likely need to create smaller labels or to add the labels after expansion was complete.

They are also exploring whether other types of polymers, or modified versions of the tetra-gel polymer, could help them realize greater accuracy. If they can achieve accuracy down to single molecules, many new frontiers could be explored, Boyden said. “If you could see individual molecules and identify what kind they are, with single-digit-nanometer accuracy, then you might be able to actually look at the structure of life. And structure, as a century of modern biology has told us, governs function,” he added.

For example, scientists could glimpse how different molecules interact with each other, which could shed light on cell signaling pathways, immune response activation, synaptic communication, drug-target interactions, and many other biological phenomena.

“We’d love to look at regions of a cell, like the synapse between two neurons, or other molecules involved in cell-cell signaling, and to figure out how all the parts talk to each other,” Boyden said. “How do they work together and how do they go wrong in diseases?”

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