Table 1.

Advantages and Disadvantages of Each Imaging Modality for Collagen Visualization.

	SHG	PSR-POL
Contrast mechanism	Intrinsic signal from light interaction with non-centrosymmetric fibrillar collagen structure	Collagen affinity for Sirius Red F3B
Specificity	Direct visualization of fibrillar collagens (ie, types I, III). Also sensitive to other harmonophores (actomyosin, microtubules)	PSR dye alone is not specific for collagen, but when paired with polarized light, fibrillar collagens undergo specific enhancement in birefringence enabling visualization
Equipment	Extensive equipment	Standard transmission pathology scope with two accurately crossed polarization filters (polarizer and analyzer)
Cost	Expensive	Inexpensive
Resolution	Easier to visualize individual collagen fibers. Forward–backward scattering ratio	Easier to visualize bundles of collagen. Detected straightness and width significantly higher
Utility	No staining required and can be done on unlabeled sections making retrospective studies possible. Submicron resolution allows visualization of individual fibers. Forward–backward scattering ratio. Can optically section thick (500–1000 µm) tissues	Requires staining of sections which might not be possible for retrospective studies. Resolution limited by visible light illumination. Can visualize counterstained cell structures directly without polarizer in place enabling examination of the stroma relative to a boundary

Abbreviations: SHG, second harmonic generation; PSR-POL, picrosirius red stain with standard polarization microscopy; PSR, picrosirius red.