Precision Histology: Quantitative Masson’s Trichrome

What is Masson’s Trichrome?

Masson’s Trichrome is one of the most common histological stains used to distinguish between muscle and collagen in tissue samples. While the original formulation has changed over the years, Masson’s Trichrome remains a three-dye based protocol that selectively stains muscle, erythrocytes and cytoplasm red, collagen blue and nuclei dark brown (Figure 1).

Figure 1: Liver tissue stained with Masson’s Trichrome.

Fibrosis is characterized by excessive formation of fibrous connective tissue, generally initiated by inflammatory responses or tissue damage. Collagen is the main component of connective tissue; therefore Masson’s Trichrome staining of increased collagen accumulation in tissue is an accurate indicator of fibrosis. Numerous diseases affecting various tissues feature fibrosis, including non-alcoholic steatohepatitis (NASH), chronic kidney disease, Crohn’s disease, cystic fibrosis, cardiac fibrosis, muscular dystrophy and many others. Thus, Masson’s Trichrome stain has become the preferred method for histopathological identification of fibrosis in both patient biopsies and animal models of human fibrotic disease.

Quantifying Masson’s Trichrome

Figure 2: Image data of Masson’s Trichrome staining of mouse liver (left) and Trichrome-positive region after tissue structure masking (right).

Although Masson’s Trichrome is routinely used to observe fibrosis, quantification of fibrosis in biological samples is largely a labor-intensive manual process that is prone to variability and bias. Therefore, there is a need for sensitive imaging tools to accurately measure fibrotic area in tissue samples. To address this need, our scientists at Reveal Biosciences have developed an AI-based, automated quantification method using our proprietary imageDx™ software that specifically isolates the Trichrome positive region within a whole tissue section.

After whole slide scanning of the Trichrome stained tissue, the image is processed using two different masks. The first mask excludes any airspace and background, while the second mask excludes the tissue structure, resulting in the isolation of the Trichrome signal (Figure 2).

After the Trichrome signal is isolated, automated tissue analysis is performed to generate insights from the tissue such as area & percentage of Trichrome-positive tissue, fibrosis intensity, and fibrosis junction branch analysis.

AI-powered quantification of Masson’s Trichrome staining using imageDx™ significantly reduces turnaround time and avoids the variability associated with traditional manual methods. You provide either whole slide images, stained slides, or fixed tissue, and our expert team will generate detailed fibrosis insights from your Masson’s Trichrome-stained tissue.

For more information on how our quantitative Masson’s Trichrome technology can advance your projects, please contact us at for a quote.

By: Caroline Morel, PhD

Pathology Revealed

Sign up to receive updates on the latest AI-powered pathology breakthroughs, access digital pathology resources, and more.