Unprecedented views of the interior of cells and other nanoscale structures are now possible thanks to innovations in expansion microscopy.

The advancements could help provide future insight into neuroscience, pathology, and many other biological and medical fields.

Collaborators from Carnegie Mellon University, the University of Pittsburgh and Brown University describe new protocols for dubbed ‘Magnify’.

Yongxin (Leon) Zhao, the Eberly Family Career Development Associate Professor of Biological Sciences, said: “Magnify can be a potent and accessible tool for the biotechnology community.”

Zhao’s Biophotonics Lab is a leader in the field of enabling super-resolution imaging of biological samples through physically expanding samples in a process known as expansion microscopy.

Through the process, samples are embedded in a swellable hydrogel that homogenously expands to increase the distance between molecules allowing them to be observed in greater resolution. This allows nanoscale biological structures that previously could only be viewed using expensive high-resolution imaging techniques to be seen with standard microscopy tools.

Magnify is a variant of expansion microscopy that allows researchers to use a new hydrogel formula, invented by Zhao’s team, that retains a spectrum of biomolecules, offers a broader application to a variety of tissues, and increases the expansion rate up to 11 times linearly or ~1,300 folds of the original volume.

Zhao said: “We overcame some of the longstanding challenges of expansion microscopy.

“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.”

See also: Glowing protein improves deep-tissue images

empty gel

Zhao said that keeping different biological components intact matters because previous protocols required eliminating many various biomolecules that held tissues together. But these molecules could contain valuable information for researchers.

He stated: “In the past, to make cells really expandable, you need 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.”

With the new method, the molecules are kept intact, and multiple types of biomolecules can be labelled in a single sample.

Zhao explained: “Before, it was like having single-choice questions: if you want to label proteins, that would be the version one protocol; if you want to label nuclei, then that would be a different version; if you wanted to do simultaneous imaging, it was difficult.

“Now with Magnify, you can pick multiple items to label, such as proteins, lipids and carbohydrates, and image them together.”

standard microscopes

Lab researchers Aleksandra Klimas, a postdoctoral researcher and Brendan Gallagher, a doctoral student, were first co-authors on the paper.

Klimas said: “This is an accessible way to image specimens in high resolution.

“Traditionally, you need expensive equipment and specific reagents and training. However, this method is broadly applicable to many types of sample preparations and can be viewed with standard microscopes that you 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 could benefit from adopting the Magnify as part of their toolkits.

He said: “One of the key concepts that we tried to keep in mind was to meet researchers where they are and have them change as few things in their protocols as possible.

“It works with different tissue types, fixation methods and even tissue that has been preserved and stored. It is very flexible, in that you don’t necessarily need to redesign experiments with Magnify in mind completely; it will work with what you have already.”

The research is published in the journal Nature Biotechnology.

Image: Example 3d image of human tissues: Uterus (expansion factor 8×). Magenta, DAPI; green, ATIPF; blue, cytokeratin pan type I/II. Zoomed in regions indicated by dashed white box. Scale bars (yellow indicates post-expansion images): 5μm. Scale bars are all in biological scale. © Carnegie Mellon University.