Fig. 2.

Representative two-photon excitation fluorescence (TPEF) images of the dermis of PXE patients where we illustrate how calcification area (CA) and calcification density (CD) were calculated. For each patient, ten field of views (FOV) were selected, with an area of 420 × 420 μm². In case of each FOV, TPEF channel was analyzed after subtraction of second-harmonic generation (SHG) signal. a, e TPEF signal of representative FOVs after the subtraction of the SHG channel. b, f Areas of calcification were manually outlined using the ImageJ v1.46 software (NIH, Bethesda, MD, USA) to calculate CD. c, d, g–h Manual thresholding was applied on outlined regions of interest (ROI) to determine CD. Different CA and CD values were found in the study population. CA (23.3 ± 16.15, mean ± SD) and CD (51.6 ± 22.1, mean ± SD) showed a high SD within our PXE cohort. a–d Representative images for PXE-affected skin with relatively higher CA and lower CD. e–h Representative images for PXE-affected skin with relatively lower CA and higher CD. i–k Counting of elastic fiber number and length. i Original TPEF image, j TPEF image after the subtraction of the SHG channel, that was used to manually outline and number elastic fibers. k Number of elastin fibers in PXE patients was 40.8 ± 27.7 μm (mean ± SD) per FOV, length of elastin fibers was 32.3 [25.4 – 34.9] μm (median [interquartile range]). Image size: a, b, e, f, i–k 420 × 420 μm²; c, d, g, h 101 × 101 μm². SD, standard deviation