A convenient method for quantifying collagen fibers in atherosclerotic lesions by ImageJ software

Background and aim: Collagen fibers are the main component of atherosclerotic lesions and should therefore be used as an index to evaluate plaque stability. Although Masson’s trichrome stainis usually employed to identify the extracellular matrix, collagen fibers identified using this staining method are difficult to analyze because the three main colors often co-localize in same area within the atherosclerotic lesion. To solve this problem, we presented a convenient method for quantification of the collagen fibers in atherosclerotic lesions with Masson’s trichrome stain. Methods: This method is based on the open-resource image software ImageJ and an associated color deconvolution plugin. Original images of atherosclerotic lesions were converted into RGB images, and these images were deconvolved by ImageJ using the color deconvolution plugin. Results: The resulting monochromatic image obtained showed collagen fibers in maximal separation from background tissues. To calculate the area of the green component, collagen fibers were quantified in an accurate and efficient manner. Conclusions: Because of the simplicity and accuracy of this method, it may be widely applied for studying atherosclerosis in humans or various animal models.