Mueller polarimetric imaging of biological tissues: classification in a decision-theoretic framework.

Mueller polarimetry is increasingly recognized as a powerful modality in biomedical imaging. Nevertheless, principled statistical analysis procedures are still lacking in this field. This paper presents a complete pipeline for polarimetric bioimages, with an application to ex vivo cervical precancer detection. In the preprocessing stage, we evaluate the replacement of pixels by superpixels. In the analysis stage, we resort to decision theory to select and tune a classifier. Performances of the retained classifier are evaluated. Decision theory provides a rigorous and versatile framework, allowing generalization to other pathologies, to other imaging procedures, and to classification problems involving more than two classes.

Ex vivo Mueller polarimetric imaging of the uterine cervix: a first statistical evaluation.

Early detection through screening plays a major role in reducing the impact of cervical cancer on patients. When detected before the invasive stage, precancerous lesions can be eliminated with very limited surgery. Polarimetric imaging is a potential alternative to the standard screening methods currently used. In a previous proof-of-concept study, significant contrasts have been found in polarimetric images acquired for healthy and precancerous regions of excised cervical tissue. To quantify the ability of the technique to differentiate between healthy and precancerous tissue, polarimetric images of seventeen cervical conization specimens (cone-shaped or cylindrical wedges from the uterine cervix) are compared with results from histopathological diagnoses, which is considered to be the "gold standard." The sensitivity and specificity of the technique are calculated for images acquired at wavelengths of 450, 550, and 600 nm, aiming to differentiate between high-grade cervical intraepithelial neoplasia (CIN 2-3) and healthy squamous epithelium. To do so, a sliding threshold for the scalar retardance parameter was used for the sample zones, as labeled after histological diagnosis. An optimized value of ∼83% is achieved for both sensitivity and specificity for images acquired at 450 nm and for a threshold scalar retardance value of 10.6 deg. This study paves the way for an application of polarimetry in the clinic.

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

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.

Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging.

We studied the azimuthal orientations of collagen fibers in histological slides of uterine cervical tissue by two different microscopy techniques, namely Mueller polarimetry (MP) and Second Harmonic Generation (SHG). SHG provides direct visualization of the fibers with high specificity, which orientations is then obtained by suitable image processing. MP provides images of retardation (among other polarimetric parameters) due to the optical anisotropy of the fibers, which is enhanced by Picrosirius Red staining. The fiber orientations are then assumed to be those of the retardation slow axes. The two methods, though fully different from each other, provide quite similar maps of average fiber orientations. Overall, our results confirm that MP microscopy provides reliable images of dominant fiber orientations at a much lower cost that SHG, which remains the "gold standard" for specific imaging of collagen fibers using optical microscopy.