Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy

Abstract

Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the "gold standard" for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5 min, and sample thicknesses of 5 µm were sufficient in cervical and vaginal tissues.

Comparison of Picrosirius Red Staining With Second Harmonic Generation Imaging for the Quantification of Clinically Relevant Collagen Fiber Features in Histopathology Samples

“…41 PSR-POL can also be combined with fluorescence microscopy 44 or more advanced polarimetry setups 45 to gain additional information about the tissue. Although not encountered in this study, PSR-POL may also have limitations including potential reproducibility issues due to variations in staining protocols 46 and will need to be assessed in other tissues. Until further validation studies are performed, SHG remains the gold standard for assessing collagen alignment in the research setting, particularly for studies requiring deep three-dimensional intravital imaging, 4 forward–backward scattering ratios, 40 and circular versus linear polarization dependencies.…”

Filamin A regulates the organization and remodeling of the pericellular collagen matrix

“…We analyzed collagen fiber abundance and organization in sections that were stained with Picrosirius Red and imaged (Fig. 2) with polarized light microscopy (49, 50). Quantification of collagen fiber density was performed at molar furcation regions of the PL, a site of particularly high‐force loading in mammalian molar teeth of limited eruption (51).…”

A multiscale Mueller polarimetry module for a stereo zoom microscope

“…The effects of imaging resolution and collagen organization on image formation underscores the need for caution in interpreting some Mueller matrix imaging results; specific selections of resolutions and ROIs may affect the results and their interpretation. Indeed, the several studies quoted above that use polarimetry for collagen imaging [40][41][42][43] may need re-examination of the robustness of their findings in light of these FOV/resolution/resultant ROI selection considerations, and a multi-scale polarimetry module of the type described in this study may prove useful in such analysis. Thus, we posit that more research into what tissue structures appear in which tissue types and at what resolutions and in what MM images is warranted.…”

“…As such, there is interest in new methods, ideally label free, to image collagen organization and to monitor the changes in its amount and arrangement, potentially leading to discovery of new predictive and prognostic tools that can impact cancer management. MM polarimetry is one candidate; several groups have previously published on using it to detect collagen organization in tissues including human uterine [40,41], human colon [42], and rat tail tendon [43]. However, MM polarimetry is not a collagen specific modality, and its reported linear retardance metric is sensitive to any aligned asymmetric tissue microstructure (e.g., cardiomyocyte fibers in the heart [44]).…”