Scientists led by MIT’s Edward Boyden have applied the principles of expansion microscopy — a technique that Boyden’s lab introduced in 2015 — to develop an inexpensive imaging method that uses an ordinary light microscope to image at the scale of individual virus particles. Key to the method is a new type of hydrogel that maintains a more uniform configuration than that typically used in conventional expansion microscopy. In that technique, scientists embed biological samples in a hydrogel and expand them before imaging them with a microscope. In addition to individual virus particles, researchers said the advancement could allow them to eventually image single biomolecules. Imaging with that degree of accuracy could enable study of the basic molecular interactions that make life possible, said Boyden, the Y. Eva Tan Professor in Neurotechnology, a professor of biological engineering and brain and cognitive sciences, a member of MIT’s McGovern Institute for Brain Research, and a member of the Koch Institute for Integrative Cancer Research. “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,” said Boyden, senior author of the study describing the new method. “And structure, as a century of modern biology has told us, governs function.” MIT engineers have developed a new type of hydrogel that maintains a more uniform configuration, pictured here, allowing for greater precision for imaging biological samples down to a resolution of about 10 nm. Courtesy of Ella Maru Studio. Since its development, researchers have effectively used expansion microscopy to physically enlarge their samples by roughly fourfold in linear dimension prior to imaging them. The ability to do so allows them to generate high-resolution images with standard equipment. Paired with subsequent development from Boyden’s lab — methods for the molecular labeling of proteins, RNA, and other molecules present in a sample so they can be imaged after expansion — has indicated the high global demand for inexpensive nanoimaging, Boyden said. Where electron microscopy and superresolution imaging both deliver high resolution, the equipment required to deploy the methods is expensive. To further enhance the accuracy of expansion microscopy, the new work overcomes a bottleneck caused by an irregularity Boyden and his team described in a 2017 paper. Laboratory team members in that work achieved resolution of around 20 nm using expansion microscopy and a process in which they expanded samples twice before imaging them. The process relied on an absorbent sodium polyacrylate polymer assembled in a method known as free radical synthesis. Though the gels that the process assembled swell when exposed to water, their structures (and densities as a result) are not completely uniform. That quality caused slight distortions in the shape of the expanded sample, limiting the accuracy of subsequent imaging. A newly developed gel, tetra-gel, helped the team overcome that limitation by forming a more predictable structure. By combining tetrahedral PEG molecules with tetrahedral sodium polyacrylates, the team created a lattice-like structure more uniform than the free-radical synthesized sodium polyacryogels with which it previously worked. A demonstration that expanded the particles of herpes simplex virus type 1 (HSV-1) showed the accuracy of the improved approach. HSV-1 particles feature a distinct spherical shape. After expanding the particles and comparing the shapes to those obtained via electron microscopy, Boyden’s group found that the distortion was lower than that seen with previous versions of expansion microscopy. Implementation of the tetra-gel ultimately delivered an accuracy of about 10 nm. The work also involved attempts to physically expand cells using the new hydrogel, such as human kidney and mouse brain cells. Next steps will look to enhance accuracy to a point at which users will be able to image individual molecules within such cells. Doing so would first require creating smaller labels or adding to labels after performing the expansion process; the size of antibodies used to label cellular molecules are about 10 to 20 nm. Team members are also exploring whether other types of polymers or modified versions of the tetra-gel polymer could deliver increased accuracy. According to Boyden, single molecule-level accuracy would suffice to show how different molecules interact with one another. That, he said, would yield new information on cell signaling pathways, immune response activation, synaptic communication, drug-target interactions, and other biological phenomena. The research was funded by Lisa Yang, John Doerr, Open Philanthropy, the National Institutes of Health, the Howard Hughes Medical Institute Simons Faculty Scholars Program, the Intelligence Advanced Research Projects Activity, the U.S. Army Research Laboratory, the U.S.-Israel Binational Science Foundation, the National Science Foundation, the Friends of the McGovern Fellowship, and the Fellows Program of the Image and Data Analysis Core at Harvard Medical School. The research was published in Nature Nanotechnology (www.doi.org/10.1038/s41565-021-00875-7).