Expansion microscopy, in which biological samples are embedded in hydrogel that homogeneously expands to increase the distance between molecules, allows nanoscale structures to be viewed in higher resolution using standard diffraction-limited microscopes. However, expansion microscopy has several limitations, such as difficulty retaining a variety of biomolecules in the gel and challenges in expanding a wide range of tissue types by 10 times or more. Researchers from Carnegie Mellon University, the University of Pittsburgh and Brown University have now described a new protocol and novel hydrogel formulation that enable retention of a spectrum of biomolecules, improve expansion of many different tissue types and allow for expansion of conventionally preserved samples up to 11-fold.
The new technique, called Magnify, uses a hydrogel containing specific proportions of the monomers dimethylacrylamide acid (DMAA), sodium acrylate (SA) and acrylamide (AA), as well as the crosslinker N,N’-methylenebisacrylamide (Bis). The researchers found that this gel chemistry allowed for greater, low-distortion expansion of multiple tissues, including freshly preserved mouse brain slices (11-fold) and formalin-fixed, paraffin-embedded (FFPE) human kidney tissue sections (8.5-fold), when compared with other expansion methods. This composition also resulted in a mechanically sturdy gel that is less susceptible to breakage during expansion and processing. To retain a wide variety of biomolecules in the gel, the team used methacrolein to anchor the molecules to the gel during gelation. This protocol was found to improve preservation of nucleic acids, proteins and lipids, allowing for post-expansion labeling and observation of all these molecules simultaneously.
Video Credit: Carnegie Mellon University, Mellon College of Science
While most expansion microscopy protocols are optimized for brain tissue, the researchers were able to use Magnify on multiple tissues and tumor types including breast and colon samples. In one of the experiments, lung organoids with specific defects in cilia ultrastructure and function were examined using conventional microscopy equipment to demonstrate the ability of Magnify to visualize clinically relevant cilia pathology. Additionally, Magnify improves the expansion of tissues that have been fixed, preserved and stored, and the protocol can be flexibly adapted to suit specific tissues, fixation methods and applications, said first co-author Brendan Gallagher, a doctoral student at Carnegie Mellon. This research was published in Nature Biotechnology.
“Magnify can be a potent and accessible tool for the biotechnology community … We overcame some of the longstanding challenges of expansion microscopy,” said corresponding author Yongxin (Leon) Zhao, an associate professor of biological sciences at Carnegie Mellon. “One of the main selling points for Magnify is the universal strategy to keep the tissue’s biomolecules, including proteins, nucleus snippets and carbohydrates, within the expanded sample.”
The researchers hope to further develop Magnify to make it more accessible to the scientific community, according to Zhao. In addition to conventional microscopy methods, Magnify can also be combined with super-resolution optical fluctuation imaging (SOFI), improving the effective resolution from about 25 nm to 15 nm, the authors wrote.