Unprecedented views of the inside of cells and other nanoscale structures at the moment are possible because of innovations in expansion microscopy. The advancements could help provide future insight into neuroscience, pathology, and plenty of other biological and medical fields.
Within the paper “Magnify is a universal molecular anchoring strategy for expansion microscopy,” published Jan. 2 within the journal Nature Biotechnology, collaborators from Carnegie Mellon University, the University of Pittsburgh and Brown University describe recent protocols for dubbed Magnify.
Magnify is usually a potent and accessible tool for the biotechnology community.”
Yongxin (Leon) Zhao, the Eberly Family Profession Development Associate Professor of Biological Sciences
Zhao’s Biophotonics Lab is a frontrunner in the sphere of enabling super-resolution imaging of biological samples through physically expanding samples in a process generally known as expansion microscopy. Through the method, samples are embedded in a swellable hydrogel that homogenously expands to extend the space between molecules allowing them to be observed in greater resolution. This permits nanoscale biological structures that previously only could possibly be viewed using expensive high-resolution imaging techniques to be seen with standard microscopy tools.
Magnify is a variant of expansion microscopy that enables researchers to make use of a recent hydrogel formula, invented by Zhao’s team, that retains a spectrum of biomolecules, offers a broader application to a wide range of tissues, and increases the expansion rate as much as 11 times linearly or ~1,300 folds of the unique volume.
“We overcame a few of the longstanding challenges of expansion microscopy,” Zhao said. “One among the important selling points for Magnify is the universal technique to keep the tissue’s biomolecules, including proteins, nucleus snippets and carbohydrates, throughout the expanded sample.”
Zhao said that keeping different biological components intact matters because previous protocols required eliminating many different biomolecules that held tissues together. But these molecules could contain invaluable information for researchers.
“Up to now, to make cells really expandable, it is advisable to use enzymes to digest proteins, so ultimately, you had an empty gel with labels that indicate the situation of the protein of interest,” he said. With the brand new method, the molecules are kept intact, and multiple forms of biomolecules could be labeled in a single sample.
“Before, it was like having single-choice questions. If you would like to label proteins, that may be the version one protocol. If you would like to label nuclei, then that may be a unique version,” Zhao said. “Should you desired to do simultaneous imaging, it was difficult. Now with Magnify, you’ll be able to pick multiple items to label, comparable to proteins, lipids and carbohydrates, and image them together.”
Lab researchers Aleksandra Klimas, a postdoctoral researcher and Brendan Gallagher, a doctoral student, were first co-authors on the paper.
“That is an accessible technique to image specimens in high resolution,” Klimas said. “Traditionally, you wish expensive equipment and specific reagents and training. Nevertheless, this method is broadly applicable to many forms of sample preparations and could be viewed with standard microscopes that you just would have in a biology laboratory.”
Gallagher, who has a background in neuroscience, said their goal was to make the protocols as compatible as possible for researchers who may benefit from adopting the Magnify as a part of their tool kits.
“One among the important thing concepts that we tried to take into account was to fulfill researchers where they’re and have them change as few things of their protocols as possible,” Gallagher said. “It really works with different tissue types, fixation methods and even tissue that has been preserved and stored. It is extremely flexible, in that you just don’t necessarily need to revamp experiments with Magnify in mind completely; it’s going to work with what you could have already.”
For researchers comparable to Simon Watkins, the founder and director of the Center for Biologic Imaging on the University of Pittsburgh and the Pittsburgh Cancer Institute, the indisputable fact that the brand new protocol is compatible with a broad range of tissue types -; including preserved tissue sections -; is vital. For instance, 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.
“To illustrate you could have a tissue with dense and non-dense components, this gets around tissues that previously would not expand isometrically,” Watkins said. “Leon has been working hard on this to make this protocol work with tissues which have been archived.”
Xi (Charlie) Ren, an assistant professor of biomedical engineering at Carnegie Mellon, studies the lung tissue and tips on how to model its morphogenesis and pathogenesis. A part of his research involves researching the motile cilia that function to clear mucus within the human conducting airway. At 200 nanometers in diameter and just a couple of micrometers in length, the structures are too small to see without time-intensive technology comparable to electron microscopy. Working in collaboration with Zhao’s lab, Ren’s team developed and delivered lung organoid models with specific defects in cilia ultrastructure and performance to validate the power of Magnify to visualise clinically relevant cilia pathology.
“With the newest Magnify techniques, we will expand those lung tissues and begin to see some ultrastructure of the motile cilia even with an everyday microscope, and it will expedite each basic and clinical investigations” he said.
The researchers also were in a position to view defects in cilia in patient-specific lung cells known to have genetic mutations.
“The lung tissue engineering community at all times needs a greater technique to characterize the tissue system that we work with,” Ren said. He added that this work is a vital first step and he hopes the collaborative work with Zhao’s lab will further be refined and applied to pathology samples present in tissue banks.
Finally, the hydrogel utilized in Magnify and developed within the Zhao lab is more robust than its predecessor, which was very fragile, causing breaks in the course of the process.
“We hope to develop this technology to make it more accessible to the community,” he said. “There are different directions this could go. There’s loads of interest in using this type of tissue expansion technology for basic science.”
Alison Barth, the Maxwell H. and Gloria C. Connan Professor within the Life Sciences at Carnegie Mellon, studies synaptic connectivity during learning. She said the broad applications provided by the brand new methods can be a boon for researchers.
“The brain is an awesome place to make the most of these super-resolution techniques,” said Barth, who collaborates with the Zhao Lab on several studies. “Microscopy methods can be useful for synaptic phenotyping and evaluation across different brain conditions.
“One among the key advances on this paper is the tactic’s ability to work on many differing types of tissue specimens.”
Additional 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 Veterans Administration; and Steven L Brody and Amjad Horani of Washington University.
Source:
Carnegie Mellon University
Journal reference:
Klimas, A., et al. (2022) Magnify is a universal molecular anchoring strategy for expansion microscopy. Nature Biotechnology. doi.org/10.1038/s41587-022-01546-1.