Macro-based collagen quantification and segmentation in picrosirius red-stained heart sections using light microscopy

Figures

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  Figure 1.. Optional visualization images put out by the macro. Segmentation by concentric wall layers (A), laterolateral composition (B), or both combined (C). Crosshatching shows overlap of regions. Darker areas in (C) indicate right heart, medium areas septum, and light ones left heart. The sketches are representations of the section shown in Fig. 2B. Labeling of ventricles in sketches is done automatically by QuantSeg. False-color representation (D) highlighting collagen in the heart section shown in Fig. 2A. Rectangle represents zoomed inlay. Scale bar: 1000 μm. Abbreviations: LV: left ventricle, RV: right ventricle.

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  Figure 2.. Representative PSR-stained heart sections of 8-week-old animals. Jup-KO mouse heart displays extensive fibrosis and myocardial atrophy (A). WT control mouse heart tissue without fibrosis, representing the aspect of a healthy heart with normal collagen content (B). Stitched overviews. Scale bars: 1000 μm. Rectangles represent zoomed inlays. Abbreviations: See Fig. 1.

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  Figure 3.. Scatter plots showing collagen content determined using the conventional fluorescence technique compared to the QuantSeg macro. The same hearts from 6–8-week-old animals were analyzed using both methods. Collagen Score (A) and Area Fraction (B) represent both methods’ main output parameters. Statistics performed by two-tailed unpaired t-test with (A) and without (B) Welch’s correction. n = 9.

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  Figure 4.. Scatter plots showing additional QuantSeg output parameters. Significant differences in Total Collagen Area (A), Mean Collagen Spot Area (D), and Number of Collagen Spots (E). No differences in Mean (B) and Maximum (C) Collagen Intensity and Tissue Density (F). The same hearts from 6–8-week-old animals were analyzed. Linear axes were used in (B, C). Statistics performed by Mann–Whitney test (A) or two-tailed unpaired t-test with (D) or without (B, C, E, F) Welch’s correction. n = 9.

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  Figure 5.. Scatter plots showing data from segmented collagen quantification. PSR-stained heart sections from 6–8-week-old animals were segmented in two dimensions [latero-lateral composition (A), strata (B)] and analyzed using the macro. Statistics were performed by two-way ANOVA and Šidák’s multiple comparisons test. n = 9. Abbreviations: EP: epicardium, SEP: subepicardial layer, M: myocardium, SEN: subendocardial layer, EN: endocardium, (r): right ventricle, (s): septum, (l): left ventricle.

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  Figure 6.. Representative PSR-stained rat MI and sham heart sections. 8 weeks after MI rat hearts displayed strong, segmental, left-ventricular fibrosis and myocardial atrophy (A). 8 weeks after sham procedure, control rats represented healthy hearts with normal collagen content (B). Stitched overviews. n = 3. Scale bars: 2500 μm. Abbreviations: See Fig. 1.

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  Figure 7.. Collagen quantification of MI rat hearts with and without prior segmentation. Hearts from rats with and without myocardial infarction were examined using the QuantSeg macro. Scatter plot shows overall collagen content of MI vs. sham hearts (A). Statistics performed by two-tailed unpaired t-test. Line plots show data from collagen quantification segmented by latero-lateral composition (B). Statistics were performed by two-way ANOVA and Šidák’s multiple comparisons test. n = 3. Abbreviations: See Fig. 5.