Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light

Collagen plays a major role in the structural organization of the heart and therefore direct visualization of collagen fibers is a crucial component of cardiac analysis. Although linearly polarized light has proven an effective tool for the examination of myocardial collagen in histologic sections, the use of circularly polarized light may offer advantages and additional possibilities. We examined the potential enhancement of collagen analysis using circularly polarized light in two ways. We first measured the brightness, and hence indirectly assessed the birefringence, of collagen fibers in scars examined at different times after myocardial infarction. Secondly, we measured collagen content in myocardial tissue and compared results obtained from brightfield analysis of trichrome stained sections with those obtained from circularly polarized light analysis of picrosirius red stained sections. We observed a progressive increase in the maximum brightness of collagen fibers in the scar with time, and a time-dependent shift in the relative distribution of collagen fiber brightness from lower to higher levels. We found consistently lower values of collagen content in trichrome stained versus picrosirius red stained tissue, and concluded that trichrome staining underestimated collagen content. The information provided by these studies could not be obtained by brightfield analysis and could be only partially obtained from linearly polarized light analysis. Thus, analysis using circularly polarized light has the ability to enhance histologic assessment of tissue and can provide additional insights into the composition and structure of myocardial collagen.