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

Figures

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  Fig. 1. A diagram of quantitative collagen volume fraction (CVF) analysis from the picrosirius red-stained slice. The total myocardial area (a) and collagen area marked with black (b) were obtained semi-automatically using ImageJ software. Panel (c) shows the red collagen alone. The histological CVF was then calculated as the percentage of collagen area divided by the total myocardial area. Scale bars: 500 µm

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  Fig. 2. Visualization of myocardial microstructure. Examples of low-magnification (top, 20 ×) and high-magnification (bottom, 63 ×) images from MPM and picrosirius red staining. (a) TPEF image; (b) SHG image; (c) merging of SHG and TPEF images; (d) picrosirius red-stained image; (e–h) magnified MPM and picrosirius red-stained images of the white and black boxed regions, respectively. Cardiomyocyte (red) and myocardial fibrosis (green) could be clearly visualized by MPM. Scale bars: 500 µm in low-magnification images (top) and 25 µm in high-magnification images (bottom), MPM, multiphoton microscopy; TPEF, two-photon excited fluorescence; SHG, second-harmonic generation

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  Fig. 3. Representative images of different myocardial fibrosis types. (a–c) TPEF images; (d–f) SHG images; (g–i) merging of TPEF and SHG images; (j–l) corresponding images of picrosirius red staining. Examples shown in the figure correspond to interstitial fibrosis (top), a combination of interstitial and replacement fibrosis (middle), and replacement fibrosis (bottom), which reflect mild (10.92%), moderate (22.2%), extensive (54.51%) of MPM-derived CVF (g–i) and corresponding 9.82%, 21.4%, 57.56% of staining-derived CVF (j–l), respectively. MPM-derived CVF was comparable with staining-derived CVF. Scale bars: 500 µm for all images, CVF, collagen volume fraction; other abbreviation as in Fig. 2

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  Fig. 4. Comparisons of CVF based on different measurement modalities and cardiomyopathies. The CVF measured by MPM was comparable to that measured by picrosirius red staining, regardless of the type of cardiomyopathies (a). The CVF in ICM patients was slightly higher compared with that in DCM patients, regardless of the measurement modalities (b) ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy; other abbreviation as in Fig. 3

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  Fig. 5. Correlations between MPM-derived CVF and staining-derived CVF. MPM-derived CVF correlated well with staining-derived CVF in all samples analysis of explanted hearts (n = 120, a). Based on subgroup analysis, correlations remained favorable in ICM samples (n = 60, b) and DCM samples (n = 60, c), Abbreviation as in Fig. 4