Fig. 1.

Workflow of epidermis recognition and counter-staining based validation of strata prediction.

Cryosections of human skin biopsies were stained by immunofluorescence for the keratinocyte differentiation markers keratin 10 (K10, only expressed in suprabasal, differentiating KC), keratin 14 (K14, highly expressed in basal KC) and nuclei were stained with Hoechst dye. Whole tissue sections were scanned in fluorescent and brightfield modalities with a TissueFAXS slide scanning microscope at 20-fold magnification and subsequently processed with “StrataQuest” image analysis software. (A) Hoechst reagent stained human abdominal skin section and (B) brightfield micrographs were used as seed images for tissue prediction with StrataQuest software. Using the nuclear fluorescence channel, nuclei were automatically detected. (A, green contours). The engine “Label Graph from Coded v3” was used to connect nuclei that are in close proximity, empty spaces between the nuclei in proximity were filled using the “Morphological – BWFillHoles” engine and “nuclei map” image (C) was generated using the “Morphological – BWAreaOpen” engine. On the brightfield (“Tran channel”) image (B), a “Filter Gauss” engine was used and thresholded (“Total Area Measurements” engine). Holes were filled (“Morphological – BWFillHoles” and “Morphological – close (dilate, erode)” engines), small clusters were removed (“Morphological – BWAreaOpen” engine) resulting in “bright field area” (D). The “nuclei map” was used as a seed and grown on the “bright field area” image (E). The resulting image is the “automated prediction” (F) of the epidermis. Manual correction was applied to correct the automatic process for the “final prediction image” (G). The basal stratum (H, pink contour) was grown on the nuclei-denser edge of the epidermis based on its distance from the border. The same growth step was applied for the low suprabasal strata (I, orange contour) with the basal layer as starting point. The stratum corneum (J, red contour) was grown from the edge of the outside of the epidermis down to the edge of the “nuclei density” map. The high suprabasal strata (J, green contour) were added between the stratum corneum and the low suprabasal stratum. (K–N) IF staining for keratin 10 (K10 stained in white in H, red in K) and for keratin 14 (K14 stained white in I, green in K). Histogram presentations of abundance vs staining intensity of the predicted cells positive for K10 (L) and K14 (M) in the respective predicted strata. The median of the individual populations is indicated by dashed vertical lines. (N) Scatter plot (K10/K14) of predicted cells in basal (pink) and low suprabasal (orange) strata. The density of the different point clouds is indicated by the greyscale color bar and the outlines delimit the 95% density distributions of the respective strata. (O) Epidermal and strata prediction on a continuous stretch of a representative normal human skin tissue section (Size bars: 50 μm, except in O: 100 μm).