This image shows a stained kidney sample imaged on a two-photon microscope with blue representing nuclei and red cytoplasm. [Cody Narciso and Victoria Zellmer/University of Notre Dame]
This image shows a stained kidney sample imaged on a two-photon microscope with blue representing nuclei and red cytoplasm. [Cody Narciso and Victoria Zellmer/University of Notre Dame]

Pathologists use tissue biopsy as the gold standard for definitively diagnosing an array of diseases, such as cancer, kidney disease, or inflammatory disorders like Crohn’s disease. Typically, physicians can only take two-dimensional (2D) snapshots of the tissue, and they are limited in their ability to measure protein levels that might better explain a differential diagnosis.

Now, researchers at the University of Notre Dame have developed a new method to acquire comprehensive three-dimensional mappings of biopsied tissue that provide much more information, incorporating both data on the tissue structure and its molecular profile.

“Sometimes, it's not obvious from a single 2D slice whether there's local metastasis taking place,” remarked senior study author Jeremiah Zartman, Ph.D., assistant professor in the department of chemical and biomolecular engineering at the University of Notre Dame. “Having additional three-dimensional information could make things less ambiguous in some cases.”

Traditional pathology methods employ thin, two-dimensional slices of the tissue and then incorporate hematoxylin and eosin (H&E) to stain the cells. Hematoxylin turns nuclei blue and eosin turns other cellular components pink, enabling doctors to see the cellular structure and identify signs of disease, like cancer. Yet, this technique has its limitations. A flat, 2D image does not reveal anything about the shape and curvature of the tissue, and the process unavoidably destroys some of the sample. Moreover, there often isn't much of a particular sample to work with; physicians are typically parsimonious with the samples because biopsies can be painful for the patient.

Additionally, two-dimensional slices can also limit how much information is extracted. For instance, if researchers want to identify the location of specific proteins by tagging them with a dye, they can only do so a few at a time, due to the limitations of fluorescence microscopes. If they want data on additional proteins, they would need yet another slice.

The new technique developed by the Notre Dame researchers can avoid most of the shortcomings of traditional methods through a microfluidic approach, in which the sample is put inside a tiny chamber on a clear chip not much bigger than a dime. The enclosure lets the tissue maintain its shape and structure, which enables multiple rounds of staining and imaging. Narrow channels allow chemical solutions to be injected into the tissue.

“We demonstrate that this method preserves tissue architecture for multiple murine organs by comparing traditional 2D slices to an optically sectioned 3D H&E-mimic,” the authors wrote. “The H&E-mimic slices show a close qualitative match to traditional H&E. The 3D spatial and molecular information obtainable from this method significantly increases the amount of data available for evaluating both tissue morphology and specific biomarkers in a wide range of both research and clinically driven applications and is amenable to automation.”

The findings from this study were published recently in Biomicrofluidics in an article entitled “On-chip three-dimensional tissue histology for microbiopsies.”

Investigators can use fluorescent dyes to tag specific protein biomarkers for various disease states. Additionally, the sample is preserved in the chip, allowing researchers to remove previously used dyes through quenching processes and then tag an entirely new set of markers.

Interestingly, the chip can also provide long-term storage of the sample, allowing for analysis at a much later time. Also, its compact size requires smaller amounts of chemicals, which could potentially cut costs.

The researchers admit that while some parts of their technique are not necessarily new, its uniqueness stems from consolidation. “It's combining different approaches into a single platform in order to enable three-dimensional imaging of microbiopsies providing a rich data set,” Dr. Zartman noted.

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