Light microscopes are usually used for pathology, but fine details of cells can’t be seen with them. The new technique, developed by Harvard Medical School and MIT, relies on expansion microscopy which expands a tissue sample to 100 times its original volume before imaging it. Using this method, researchers can see features with a conventional light microscope that can normally only be seen with expensive, high-resolution electron microscopes. It also gives insight into the molecular information that electron microscopes don’t have.
“It’s a technique that could have a very broad application,” said Edward Boyden, who created the expansion microscopy method, in a press release.
Boyden and his colleagues have used the method to distinguish early-stage breast lesions that have a high or low risk of developing cancer. The researchers were able to analyze kidney tissue and determine signs of kidney disease, suggesting that the method could be used for other diseases.
“Using expansion microscopy, we are able to diagnose diseases that were previously impossible to diagnose with a conventional light microscope,” said Octavian Bucar, one of the study’s lead authors and an instructor at Harvard Medical School, Beth Israel Deaconess Medical Center (BIDMC) and the Ludwig Center at Harvard.
The expansion microscopy technique embeds tissue samples in a dense, evenly generated polymer that swells up when exposed to water. Researchers anchor the polymer gel to the molecules that they need to image before the swelling happens and digests other proteins that hold tissue together.
This technique allows researchers to get images with a resolution of about 70 nm.
Researchers on this study wanted to use the expansion process to biopsy tissue samples that are usually in paraffin wax, flash froze or stained with a chemical to make cell structures more visible. The new process converts the samples into something that can be expanded.
To expand the samples, researchers remove the chemical stain or paraffin by exposing tissues to the chemical solvent known as xylene. The sample gets heated up in another chemical called citrate and the tissues go through an expansion process that is similar to the original technique, but has stronger digestion steps to make up for the strong chemical fixation of samples.
“The work of Zhao et. al. describes a very clever way of extending the resolution of light microscopy to resolve detail beyond that seen with conventional methods,” said David Rimm, a professor of pathology at Yale University’s School of Medicine.
The researchers tested their method using tissues from patients who had early-stage breast lesions. Their intentions were to predict whether the lesions will be malignant by evaluating the nuclei of the cells. Atypical nuclei in benign lesions have a fivefold higher chance of becoming cancer than typical nuclei.
Once the tissue samples were expanded, the research team analyzed them using a machine learning algorithm that rated the nuclei based on orientation, diameter, how much they deviate from true circularity and more. The algorithm is designed to differentiate between invasive and non-invasive lesions within 93% accuracy on expanded samples while pre-expanded tissue had an accuracy of 71%.
They also used kidney tissue samples from patients who had nephrotic syndrome. Pathologists were able to identify diseased tissue on expanded tissues with 90% accuracy and only 65% accuracy on unexpanded tissue samples.
Researcher hope that this process could be further developed to create more precise diagnostics for other diseases.
“Cancer biopsies are just the beginning,” said Boyden. “We have a new pipeline for taking clinical samples and expanding them, and we are finding that we can apply expansion to many different diseases. Expansion will enable computational pathology to take advantage of more information in a specimen than previously possible.”
The research was funded by the New York Stem Cell Foundation Robertson Investigator Award, the National Institutes of Health Director’s Pioneer Award, the Department of Defense Multidisciplinary University Research Initiative, the Open Philanthropy Project, the Ludwig Center at Harvard and Harvard Catalyst. It was published online in the journal Nature Biotechnology.
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