Researchers have created a method to enlarge tissue samples by 20 times in one go. This straightforward and affordable approach could enable almost any biology lab to conduct nanoscale imaging.
Traditionally, observing nanoscale structures within cells requires high-end, costly super-resolution microscopes. In contrast, MIT investigators have introduced a technique that enables the expansion of tissue prior to imaging, achieving nanoscale clarity with a regular light microscope.
In their latest advancement, the researchers have achieved a remarkable 20-fold tissue expansion in one step. This uncomplicated and low-cost technique has the potential to allow nearly every biology lab to carry out nanoscale imaging.
“This democratizes imaging,” states Laura Kiessling, the Novartis Professor of Chemistry at MIT and a member of both the Broad Institute of MIT and Harvard and MIT’s Koch Institute for Integrative Cancer Research. “Previously, to observe high-resolution images, you had to rely on very expensive microscopes. This new approach enables the visualization of structures that would typically remain unseen with standard microscopes, making imaging more affordable by eliminating the need for specialized facilities.”
At the resolution provided by this technique, approximately 20 nanometers, researchers can observe organelles within cells and groups of proteins.
“Achieving a twenty-fold expansion brings us into the scale where biological molecules function. The essentials of life, including biomolecules, genes, and their products, are nanoscale entities,” notes Edward Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT; he also serves as a professor of biological engineering, media arts and sciences, and brain and cognitive sciences; is a Howard Hughes Medical Institute investigator; and is part of MIT’s McGovern Institute for Brain Research and Koch Institute for Integrative Cancer Research.
Boyden and Kiessling are the principal authors of this new study, which will be published in Nature Methods. The paper is led by MIT graduate student Shiwei Wang and Tay Won Shin, PhD ’23.
A Single-Step Expansion
Boyden’s team created expansion microscopy back in 2015. This method involves embedding tissue in an absorbent polymer and dismantling the proteins that typically hold the tissue intact. When water is introduced, the gel swells, separating biomolecules from one another.
The previous version of this technique allowed for approximately a fourfold expansion, resulting in images with a resolution of around 70 nanometers. In 2017, Boyden’s lab adapted the method to include an additional expansion step, achieving a total 20-fold expansion for improved resolution, albeit with added complexity.
“We developed several 20-fold expansion technologies before, but they required multiple expansion phases,” Boyden explains. “If we could achieve that level of expansion in just one step, it would simplify the process significantly.”
With the 20-fold expansion, researchers can reach a resolution of roughly 20 nanometers using a standard light microscope, allowing them to visualize cell components such as microtubules and mitochondria, along with clusters of proteins.
The researchers aimed to perform the 20-fold expansion in just one step, requiring a gel that was both highly absorbent and mechanically durable to withstand the expansion process.
To accomplish this, they developed a gel from N,N-dimethylacrylamide (DMAA) and sodium acrylate. Unlike past expansion gels, which needed another molecule to create crosslinks between the polymer strands, this new gel spontaneously forms crosslinks and possesses strong mechanical properties. Previous gel components had been used in expansion microscopy techniques, but those gels could only expand about tenfold. The MIT team refined the gel and polymerization process to enhance its strength, ensuring it could expand 20 times.
To improve the gel’s stability and consistency, the researchers eliminated oxygen from the polymer solution before gelation, preventing side reactions that could disrupt crosslinking. This operation involved flowing nitrogen gas through the polymer solution, displacing most of the oxygen in the mixture.
Once the gel is formed, specific bonds within the proteins that keep the tissue intact are broken, and water is introduced to expand the gel. Following the expansion, researchers can label target proteins in the tissue and image them.
“While this method may involve more sample prep than other super-resolution techniques, the actual imaging process is considerably simpler, particularly for 3D imaging,” Shin notes. “We provide a detailed step-by-step protocol in the manuscript for readers to easily follow.”
Imaging Minuscule Structures
The team successfully used this technique to visualize various minuscule structures within brain cells, including synaptic nanocolumns—clusters of proteins arranged in a specific structure at neuronal synapses that facilitate communication between neurons through neurotransmitters like dopamine.
In their investigations of cancer cells, the researchers also used the method to observe microtubules—hollow tubes essential for maintaining cell shape and driving cell division. They further imaged mitochondria, which produce energy, and even the layout of individual nuclear pore complexes (protein clusters that regulate access to the cell nucleus).
Wang is currently applying this technique to visualize carbohydrates known as glycans, which are present on cell surfaces and play a crucial role in cell-environment interactions. This method holds the potential for examining tumor cells, offering a clearer view of protein organization within those cells, far more straightforwardly than ever before.
The researchers believe that this technique can be widely adopted in any biology lab due to its reliance on standard, readily available chemicals and common equipment, such as confocal microscopes and glove bags, which most laboratories already possess or can easily obtain.
“We hope that with this innovative technology, any standard biology lab can utilize this protocol with their existing microscopes, enabling them to reach resolutions previously only attainable with specialized and expensive state-of-the-art microscopes,” Wang remarks.
The research received funding partly from the U.S. National Institutes of Health, the MIT Presidential Graduate Fellowship, U.S. National Science Foundation Graduate Research Fellowship grants, Open Philanthropy, Good Ventures, the Howard Hughes Medical Institute, Lisa Yang, Ashar Aziz, and the European Research Council.
