Stainless Staining Provides New Histology Tool for Researchers and Clinical Pathologists

Researchers have developed a potentially revolutionary system based on infrared spectroscopic imaging that enables stain-free, non-perturbing, multiple analyses of a single biological tissue sample.

Histology staining has evolved with non-specific stains, then immunohistochemical staining, and then digital imaging with computerized analysis. In these cases, the prepared biopsy samples are stained. In the new study, a team of engineers, pathologists, and surgeon, led by Prof. Rohit Bhargava of the Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, (Urbana and Champaign, IL, USA), used a new approach, based on Fourier transform infrared (FT-IR) spectroscopic imaging and computation, to develop stain- and label-free chemical imaging to provide the same information as molecular stains. This enables a rapid, digital, quantitative, and non-perturbing visualization of morphology and multiple molecular epitopes for various applications.


The spectra measure the chemical constitution of cells and tissues directly. With computational techniques, the researchers were able to relate spectral properties to known staining patterns of tissue. Using computation and the intrinsic molecular contrast of the tissue enabled molecular stains to be digitally produced without staining the tissue itself. Thus, staining can be visualized while leaving precious tissue pristine for downstream analyses and without the added material cost, time, and labor of direct tissue staining.

This approach also enables analysis of small samples (for example from a thin needle biopsy) or where it may not be possible to obtain multiple samples from the same biopsy for multiple stains. In cases where materials are limited or there may be a need to closely correlate multiple expressed molecules, the new method could be a solution, allowing the user to simply "dial-in" a required stain.

The study is timely as it builds on the emergence of chemical imaging and maturation of computation from the science and engineering side and the drive to greater molecular content information from the biomedical, clinical side. This approach has potential for immediate and long term impact in changing the field to a multiplexed molecular science in research and clinical practice, histology and pathology.

"We're relying on the chemistry to generate the ground truth and act as the "supervisor" for a supervised learning algorithm. A human has to verify that the stain was applied appropriately, but that's something that pathologists do very well", said lead author David Mayerich, assistant professor, University of Houston. Michael Walsh, assistant professor, University of Illinois at Chicago, added, "One of the bottlenecks in automated pathology is the extensive processing that must be applied to stained images to correct for staining artifacts and inconsistencies. The ability to apply stains uniformly across multiple samples could make these initial image processing steps significantly easier and more robust".

"FT-IR imaging allows histology digital imaging without destroying tissue properties caused by staining; therefore, the same slide can be used for other purposes (multiplex immunofluorescence and immunohistochemistry, or other methods). For research applications, it also allows higher throughput by rapidly marking up the tissues for regions of interest," said Dr. Andre Balla. "Infrared and optical imaging seemed to be distinct modes for getting important data in pathology. This study shows a close link between the two, allowing the user to choose the best method to address their needs," said Prof. Bhargava.

The study, by Mayerich D. et al, was published March 20, 2015, in the journal Technology.

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