Three-Dimensional Imaging of Human Myocardial Collagen Fibers Using Trypan Blue Staining

The aim of the study was to estimate the availability of trypan blue staining of human myocardium sections to investigate cardiac collagen fibers, in particular, their three-dimensional reconstructions.

Pretreated with phosphomolybdic acid sections of human myocardium (n=9) were stained with 1% aqueous trypan blue solution (Panreac, Spain) (patent application RU 2024/22564). Parallel sections were stained using the similar technique with 2% aqueous solution of aniline blue (Unisource Chemicals Pvt. Ltd., India). The obtained preparations were analyzed using a light microscope Leica DM750 (Leica, Germany) and a confocal laser microscope LSM800 (Carl Zeiss AG, Germany). Threedimensional reconstruction was performed using software ZEN-2012 (Carl Zeiss AG, Germany).

Trypan blue appeared to stain collagen fibers selectively. Loose thick fibers were stained more intensively than thin fibers, which were not available for detailed examination with bright-field microscopy. The cytoplasm of cardiomyocytes remained unstained. Aniline blue appeared to stain thin fibers relatively more intensively, and its non-specifically stained the cytoplasm of cardiomyocytes.Fluorescence of collagen fibers stained with trypan blue was excited with a 640 nm laser. Autofluorescence of the cytoplasm of cardiomyocytes was excited with a 488 nm laser. Three-dimensional reconstruction of endomysium showed that it was a net. Therefore, it became possible to describe the lower-order structures being the part of thick fibers. Three-dimensional reconstruction enabled to distinguish the orientation of the fibers and their cross section differentiating it from tangential one.

The study findings showed trypan blue staining of human cardiac section to be an effective method to reveal collagen fibers, study their structure with bright-field microscopy using confocal laser microscopy and perform detailed three-dimensional reconstructions. The advantages of the method compared with other techniques of three-dimensional imaging of collagen fibers consist in the accessibility of reagents, high staining selectivity of the connective tissue matrix, the ability to obtain contrast and detailed images of the studied objects with high resolution.