Conventional and confocal fluorescence microscopy of collagen fibers in the heart

The arrangement of collagen fibers has previously been studied with picrosirius red (PSR) staining and brightfield microscopy. We discovered that PSR staining can also be visualized by fluorescence microscopy. PSR-stained collagen was strongly fluorescent using excitation and barrier filters for rhodamine, and distracting background cytoplasmic fluorescence was drastically reduced with phosphomolybdic acid (PMA) treatment before PSR staining. The PMA-PSR fluorescence method was more sensitive than the brightfield PSR or PMA-PSR method, and permitted confocal microscopic study. We applied the method to the study of collagen fiber three-dimensional arrangement in perimysial and endomysial septa of the heart, showing the three-dimensional course of the fibers in stereo views generated by confocal microscopy. The PMA-PSR fluorescence method should be generally useful for accurately determining collagen fiber three-dimensional arrangement, a necessary prelude to mechanical modeling of collagen-reinforced tissues.