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. 2021 Sep 16;10:e68920. doi: 10.7554/eLife.68920

Figure 2. Silver-based X-ray microtomography (micro-CT) enables segmentation and visualization of melanin microanatomy in three dimensions (3D).

(A) A light micrograph of the side of a 5 days post-fertilization (dpf) wild-type larva indicating the major regions of melanin pigment: dorsal, lateral, ventral, and yolk sac stripes and the retinal pigment epithelium (RPE). (B) A 3D rendering with orthoslice (C) of micro-CT volumes segmented into anatomical regions shows the organization of larval pigment into layers. Red = dorsal stripe, yellow = ventral stripe, green = yolk sac stripe, cyan = lateral stripes, white = RPE, gray = other body melanin, purple = lens. (C) Single slice of micro-CT data with color overlay corresponding to the indicated region in B. (D) 200-slice maximum intensity projection of dorsal stripe melanin exhibits transparencies in the staining indicating the position of large organelles (arrows) including potentially binucleated cells (circles). A sample of these transparencies was measured to estimate average size (Figure 2—figure supplement 1, Figure 2—source data 1). (E) View from the top-down of a volume rendering showing rostral melanin in the nose forming globular, dendritic cells (arrowheads). RPE-L = left RPE, RPE-R = right RPE. (F) Isolated volume rendering of the right eye with a clipping plane showing the villous inner surface and smooth outer surface of the RPE. The rendering has been falsely colored by intensity to highlight certain anatomical features, including local pigment thickness variability throughout the RPE, the egress of the optic nerve (arrowhead), and the fused choroidal fissure (arrows). L = lens.

Figure 2—source data 1. Measurements of ovoid transparencies in melanin staining.

Figure 2.

Figure 2—figure supplement 1. Measurements of ovoid transparencies in melanin staining.

Figure 2—figure supplement 1.

Volume renderings of the top of the head for three 5 days post-fertilization (dpf) wild-type zebrafish were examined for ovoid transparencies in the staining. For each fish, 25 random opacities were selected and manually measured on the long axis using the three-dimensional (3D) line measurement tool in Avizo (pink lines). Average length (n = 75) was 9.29 µm (SD = ±1.23 µm). Full list of measurements is provided in Figure 2—source data 1.