Unprecedented views of the interior of cells and other nanoscale structures are now possible thanks to innovations in expansion microscopy. The advances might help provide future insight into neuroscience, pathology, and many other biological and medical fields.
In the article “Magnify is a universal molecular anchoring strategy for expansion microscopy,” published Jan. 2 in the journal Natural biotechnologycollaborators from Carnegie Mellon University, University of Pittsburgh, and Brown University describe new protocols for Magnify.
Magnify can be a powerful and accessible tool for the biotech community. »
Yongxin (Leon) Zhao, Eberly Family Career Development Associate Professor in Biological Sciences
Zhao’s Biophotonics Laboratory is a leader in the field of super-resolution imaging of biological samples through the physical expansion of samples in a process known as expansion microscopy. Throughout the process, samples are embedded in a swellable hydrogel that expands evenly to increase the distance between molecules, allowing them to be observed with greater resolution. This makes it possible to see biological structures on the nanoscale that previously might only be visualized using expensive high-resolution imaging techniques with standard microscopy tools.
Magnify is a variation of expansion microscopy that allows researchers to use a new hydrogel formula, invented by Zhao’s team, that retains a spectrum of biomolecules, offers broader application to a variety of tissues and increases the rate of expansion up to 11 times linearly or ~1,300 folds of the original volume.
“We overcame some of the long-standing challenges in expansion microscopy,” Zhao said. “One of Magnify’s key selling points is the universal strategy of retaining tissue biomolecules, including proteins, core extracts and carbohydrates, in the expanded sample. »
Zhao said it was important to keep different biological components intact because previous protocols required the removal of many diverse biomolecules that held tissues together. But these molecules might contain valuable information for researchers.
“In the past, to make cells really expandable, you had to use enzymes to digest proteins, so in the end you had an empty gel with labels that indicate the location of the protein of interest,” said he declared. With the new method, molecules are kept intact and multiple types of biomolecules can be labeled in a single sample.
“Before, it was like having single-choice questions. If you want to label proteins, that would be version 1 of the protocol. If you want to label the cores, that would be a different version,” Zhao said. “If you wanted to do simultaneous imaging, it was difficult. Now, with Magnify, you can choose multiple items to label, such as proteins, fats, and carbohydrates, and image them together. »
Laboratory researchers Aleksandra Klimas, postdoctoral researcher and Brendan Gallagher, doctoral student, were the first co-authors of the article.
“This is an accessible way to image specimens in high resolution,” Klimas said. “Traditionally, you need expensive equipment, specific reagents and training. However, this method is widely applicable to many types of sample preparations and can be viewed with standard microscopes that you would have in a biology lab. »
Gallagher, who has a background in neuroscience, said her goal was to make the protocols as compatible as possible for researchers who might benefit from adopting the Magnify as part of their toolkits.
“One of the key concepts that we tried to keep in mind was to meet the researchers where they are and have them change as little as possible in their protocols,” Gallagher said. “It works with different tissue types, fixation methods, and even tissue that has been preserved and stored. It’s very flexible, in that you don’t necessarily need to completely redesign experiences with Magnify in mind; it will work with what you already have. »
For researchers such as Simon Watkins, founder and director of the Center for Biological Imaging at the University of Pittsburgh and the Pittsburgh Cancer Institute, the fact that the new protocol is compatible with a wide range of tissue types –; including preserved tissue sections –; is important. For example, most expansion microscopy methods are optimized for brain tissue. In contrast, Magnify was tested on samples from various human organs and corresponding tumors, including breast, brain, and colon.
“Let’s say you have tissue with dense and non-dense components, it bypasses tissue that previously didn’t expand isometrically,” Watkins said. “Leon has worked hard to make this protocol work with tissue that has been archived. »
Xi (Charlie) Ren, assistant professor of biomedical engineering at Carnegie Mellon, studies lung tissue and how to model its morphogenesis and pathogenesis. Part of his research involves looking for the motile cilia that work to clear mucus in the human airways. At 200 nanometers in diameter and only a few micrometers in length, the structures are too small to see without time-consuming technology such as electron microscopy. In collaboration with Zhao’s lab, Ren’s team developed and provided models of lung organoids with specific defects in cilia ultrastructure and function to validate Magnify’s ability to visualize clinically relevant cilia pathology. .
“With the latest Magnify techniques, we can expand these lung tissues and begin to see mobile cilia ultrastructure even with a regular microscope, which will speed up basic and clinical investigations,” he said.
The researchers were also able to visualize defects in the cilia in patient-specific lung cells known to have genetic mutations.
“The lung tissue engineering community always needs a better way to characterize the tissue system we’re working with,” Ren said. He added that this work is an important first step and he hopes that the collaborative work with Zhao’s lab will be further refined and applied to pathology samples found in tissue banks.
Finally, the hydrogel used in Magnify and developed in Zhao’s lab is sturdier than its predecessor, which was very fragile, causing breakages in the process.
“We hope to develop this technology to make it more accessible to the community,” he said. “It can go in different directions. There is a lot of interest in using this type of tissue expansion technology for basic science. »
Alison Barth, Maxwell H. and Gloria C. Connan Professor of Life Sciences at Carnegie Mellon, studies synaptic connectivity during learning. She said the broad applications provided by the new methods will be a boon to researchers.
“The brain is a great place to take advantage of these super-resolution techniques,” said Barth, who is collaborating with the Zhao Lab on several studies. “Microscopy methods will be beneficial for synaptic phenotyping and analysis in different brain conditions.
“One of the major advances of this paper is the ability of the method to work on many different types of tissue samples. »
Other study authors include Piyumi Wijesekara, Emma F. DiBernardo, Zhangyu Cheng of Carnegie Mellon; Sinda Fekir and Christopher I. Moore of Brown University; Donna B. Stolz of Pitt; Franca Cambi of Pitt and the Veterans Administration; and Steven L Brody and Amjad Horani of the University of Washington.
