Multiphoton microscopy providing pathological-level quantification of myocardial fibrosis in transplanted human heart

Multiphoton microscopy (MPM), a high-resolution laser scanning technique, has been shown to provide detailed real-time information on fibrosis assessment in animal models. But the value of MPM in human histology, especially in heart tissue, has not been fully explored. We aimed to evaluate the association between myocardial fibrosis measured by MPM and that measured by histological staining in the transplanted human heart. One hundred and twenty samples of heart tissue were obtained from 20 patients consisting of 10 dilated cardiomyopathies (DCM) and 10 ischemic cardiomyopathies (ICM). MPM and picrosirius red staining were performed to quantify collagen volume fraction (CVF) in explanted hearts postoperatively. Cardiomyocyte and myocardial fibrosis could be clearly visualized by MPM. Although patients with ICM had significantly greater MPM-derived CVF than patients with DCM (25.33  ± 12.65 % vs. 19.82  ± 8.62 %, p = 0.006), there was a substantial overlap of CVF values between them. MPM-derived CVF was comparable to that derived from picrosirius red staining based on all samples (22.58 ± 11.13% vs. 21.19 ± 11.79%, p = 0.348), as well as in DCM samples and ICM samples. MPM-derived CVF was correlated strongly with the magnitude of staining-derived CVF in both all samples and DCM samples and ICM samples (r = 0.972, r = 0.963, r = 0.973, respectively; all p < 0.001). Intra- and inter-observer reproducibility for MPM-derived CVF and staining-derived CVF were 0.995, 0.989, 0.995, and 0.985, respectively. Our data demonstrated that MPM can provide a pathological-level assessment of myocardial microstructure in transplanted human heart.