Bergmann glial cells immunolabelled with an anti-GFAP monoclonal antibody, observed in a LUCID-treated rat cerebellum.
University of Tokyo researchers have developed a method for rendering organs and tissues transparent while preserving their three-dimensional structures. Their reagent, IlLUmination of Cleared Organs to IDentify Target Molecules, or LUCID, is set to make sophisticated diagnostics more widely accessible and to change the landscape of medical research and pathology.
Developing a new way to view organs
Hiroshi Onodera, a professor of engineering at the University of Tokyo, conceptualized LUCID in 2011, when the Tohoku earthquake caused damage to his lab and delayed experiments.
“Since there was a possibility that samples under cryopreservation would deteriorate due to the power outage after the earthquake, we wondered if we could make them transparent and store them at room temperature for a long time,” he explains.
He turned to the scientific literature to continue his research, and became intrigued by so-called ‘clearing’ agents that could render biological tissues transparent, which allowed the structures within them to be seen using light microscopes.
A mouse small intestine treated with LUCID.
Cells consist of fluid and organelles surrounded by membranes. When a cell is illuminated, the organelles and fluid within them scatter light because of the differences in their refractive indices, which makes cells and tissues appear opaque.
Onodera began immersing test samples in different reagents with high refractive indices and high cell permeabilities that could homogenize this light scattering, but that were also non-carcinogenic and not damaging to the tissue itself. He discovered that a series of (2,2’-thiodiethanol)-based solutions could effectively replace the fluid inside tissues, and crucially – match its refractive index to that of the membranes, rendering the tissue transparent. Onodera patented this revolutionary method in 2014.
“Without damaging them, LUCID penetrates cells to suppress light scattering, and make tissues transparent for observation under microscopes,” explains Junji Yumoto, a professor emeritus at the University of Tokyo and a COI project leader. “It differs significantly from other tissue clearing methods, which break down the lipid membranes, and therefore compromise the structure of the tissue.”
New imaging capability for scientists
As a gentler method for rendering biological samples transparent, LUCID is a valuable tool to guide research into new therapeutic agents. Another benefit is its ability to retain fluorescent stains that are commonly used to image specific biological features, such as cancerous cells within a tissue sample, for several months.
One example of this success is outlined in a 2018 Pathology International report, which describes LUCID’s ability to reveal 3D structures of cancerous tissue within a porcine gastrointestinal specimen at higher resolution than previously possible. Such detail could help identify new targets that can be explored for therapeutic intervention.
The study also suggested the method could be clinically used to analyse biopsies obtained by endoscopy, providing a potentially more sensitive way to detect bowel cancer development and spread.
Better access for pathologists
Since 2017, Onodera’s collaborators at the University of Tokyo Hospital have used LUCID to process tumour samples for imaging. Yumoto says the reagent, if implemented more broadly, could significantly help cancer detection.
“In pathology labs, biopsies are currently sectioned into thin slices before analysis, which can allow diseased tissues and cells, such as cancer, to be overlooked. LUCID not only eliminates this sample preparation, it allows the entire sample to be observed in three dimensions using standard microscopes,” he says.
Unlike other methods, LUCID doesn’t contain toxic or flammable substances that could preclude its use for routine analysis. It has also been able to preserve samples for at least eight years, so it is compatible with Japan’s pathology regulations, which demand biopsies be stored for five years after excision, says Yumoto.
Embarking on new frontiers in biology
The transparency afforded by LUCID has enabled researchers to track the spread of cancer cells tagged with a green fluorescent protein (GFP) in a mouse model of gastric cancer. The benefit of LUCID in this case is that GFP fluorescence may be maintained even after a year, unlike other clearing methods in which fluorescence is rapidly lost over time. Researchers have also used LUCID to render a colon transparent in a mouse model of colitis, which enabled them to clarify the role of CD115+ monocytes in the disease.
After the recent formation of a venture company, LUCID is now available to researchers and pathologists at universities and hospitals globally. Meanwhile, the University of Tokyo team are continuing to refine the method for new uses. “We believe we can make any tissue transparent,” says Yumoto.