Fig. 3

a Loose iridophores in stripe region viewed by incident illumination, fluorescence, and high pressure–frozen, freeze-fractured cryo-SEM (Cryo-SEM). The incident illumination image shows blue iridophores on top of black melanophores; the fluorescent image reveals malachite green (MG) labeled iridophores (pseudo colored green) with highly ordered arrays of guanine crystals; and the Cryo-SEM image shows iridophore cytoplasm with highly disordered arrays of crystals. n = 5 adult fish for incident illumination and fluorescence, and n = 4 adult fish for cryo-SEM. b Dense iridophores in interstripe region viewed by incident illumination, fluorescence, and Cryo-SEM. The incident illumination image shows silvery iridophores covered by yellow xanthophores; the fluorescent image reveals MG-labeled iridophores with disordered arrays of guanine crystals (pseudo colored green); and the high pressure–frozen, freeze-fractured cryo-SEM micrograph shows iridophore cytoplasm with disordered arrangements of crystals. n = 5 adult fish for incident illumination and fluorescence, and n = 4 adult fish for cryo-SEM. c–e TEM analysis of crystals isolated from iridophores from either the stripe or the interstripe regions of adult fish (n = 4 adult fish). c TEM micrographs of crystals isolated from stripe (left panel) and interstripe regions (right panel); d TEM-based electron diffraction of the crystals shown in Supplementary Fig. 7, isolated from stripe iridophores (left panel) and interstripe iridophores (right panel); e graph of aspect ratio (length/width) of stripe iridophores (blue) and interstripe iridophores (orange), n = 60 for stripe isolated crystals and n = 57 for interstripe isolated crystals, Data are represented as mean ± SEM. The p value, p < 0.0001, was determined using two-tailed Mann–Whitney test. f Simulated reflection (black) and measured reflection (red) from a stripe iridophore. g Simulated reflection (black) and measured reflection (red) from an interstripe iridophore. Insets in both f and g show the corresponding reflectance color on a CIE (International Commission on Illumination) chromaticity space diagram. h Incident illumination image of an adult fish lacking melanin in melanophores and carotenoids in xanthophores due to mutations in tyrosinase and scarb1, respectively. The image shows iridophore-type-specific coloration is independent of melanin and carotenoids, consistent with reflectance data obtained for stripe (f) and interstipe iridophores (g). Scale bars, a, b (left panels) 50 μm, a, b (right panels) 4 μm, a, b (bottom panels) 1.5 μm, c 2 μm.