POLARIZED LIGHT SCANNING OF PICROSIRIUS RED WITH MVSLIDE
Picrosirius Red Staining of Collagen Fibers
Picrosirius Red (PSR) staining is based on the Sirius Red dye which is dissolved in an aqueous picric acid solution. PSR is relatively specific for collagen fibers: based on superior geometric fit and electrostatic attraction, the Sirius Red molecule binds rapidly and stably to the collagen fibers. In addition, Upon binding of Sirius Red, the birefringence of collagen fibers is enhanced, which improves their visibility and differentiation under polarized light.
Consequently, PSR-stained collagen is not only detectable under standard brightfield conditions, but also using cross-polarized light:
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In brightfield, PSR-stained collagen appears red
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Under cross-polarized light, PSR-stained thick collagen type I fibers appear yellow to orange, whereas thinner PSR-stained collagen type III fibers appear green, enabling differentiation between collagen types
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Collagen Identification and Quantification Using Whole Slide Images
The possibility of assessing total collagen in standard brightfield conditions as well as the relative distribution of collagen type I and collagen type III using cross-polarized light makes PSR one of the most frequently used stains for studying collagen networks in biological tissues. Common applications include fibrosis assessment in organisms such as liver, lung and kidney, as well as research on arterial diseases or tumor-related changes in collagen architecture.
In these contexts, the use of whole slide images is particularly important. The ability to analyze complete, representative tissue sections instead of isolated snapshots improves both workflow efficiency and the robustness of quantitative results.
The whole slide images can be analyzed using a range of existing image analysis tools. For example, the open-source platform QuPath was shown to be suitable for segmentation and quantitative assessment of PSR-stained collagen, both in brightfield images as well as in polarized light scans.
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A practical limitation in this context is that only few automated whole slide scanners support imaging under polarized light.
Polarized Light Scanning of Picrosirius Red Samples with mvSlide
mvSlide allows scanning of Picrosirius Red stained sections not only in standard brightfield but also under cross-polarized light, provided the microscope is equipped with polarization optics.
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With mvSlide, the user retains full control over focusing during acquisition. This is particularly helpful as Picrosirius Red stained collagen structures often exhibit a three-dimensional organization, for example in tissues of vascular walls or fibrotic regions. Under polarized light, and especially at higher magnifications, the limited depth of field makes accurate focusing critical, as even small deviations can significantly affect the visibility and colour appearance of birefringent fibers.
The stitching algorithm of mvSlide, combined with operator-controlled focus adjustment, enables the reconstruction of consistently well-focused whole slide images. This facilitates the creation of high-quality scans of PSR-stained collagen fibers under these demanding imaging conditions.
Example: Picrosirius Red Stained Human Radial Artery Scanned with mvSlide
(Brightfield vs Polarized Light)
Zoom into the scans using the mouse wheel to explore more image details!
Scan descriptions:
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Scan (1): Brightfield scan of a Picrosirius Red stained human radial artery section at 20X resolution acquired with mvSlide.
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Scan (2): Cross-polarized light scan of a Picrosirius Red stained human radial artery section at 10X resolution acquired with mvSlide, revealing orange type I collagen fibers and green type III collagen fibers.
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The scans were kindly provided by Julia Vettese, UCLouvain.​
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