Diffusion barriers within the medaka egg. Stage 17 embryos were incubated with 10 mg/ml fluorescein for 40 minutes at 27°C and internal concentrations were determined at different time points of washing (A; mean values of 8–10 eggs are shown +/− SEM). (A) Dotted lines indicate the bi-phasic out-diffusion: (a) fast loss of the signal (first 2 measuring points), (b) slower second phase (best-fit of last 4 measuring points). (B, left side) shows a schematic view of an egg and the consecutive diffusion steps (red arrows). (C) Bright field picture of a dead embryo, representing diffusion through the chorion; the black arrow head indicates the shrunken embryo/yolk with the surrounding membranes. (D) Dechorionated embryo at stage 22 in dorsal view with anterior being at the top, (C’, D’) corresponding fluorescent images. (E) Concentrations of fluorescein in dead or dechorionated embryos after different incubation times. The concentration of the supernatant (blue) was set to 100%. Several measuring points (7, 20 and 30 min) were taken and the initial concentration at the onset of the washing calculated by extrapolation (compare A). (F-H) Eggs in brightfield, fluorescence and merged pictures. (F-F”) Distribution of fluorescein after 10 minutes of in-diffusion (IN early) and 2 minutes of washing (1 mg/ml fluorescein). (G-G”) Out-diffusion after 40 minutes of incubation (10 mg/ml fluorescein) and 5 minutes washing (OUT early) and (H-H”) same conditions as in (G) but 30 minutes of washing time. Scale bar 250 μM.