Nano-Scale Morphology Of Melanosomes Revealed By Small-Angle ...

3.2. X-ray scattering at cryogenic temperatures

In order to obtain information complementary to that from the SAXS experiment described above, where many suspended organelles were illuminated simultaneously, and thus the specific structure of the D2 could not be disentangled with respect to surface and bulk information at low q-values, we performed a scanning experiment with single vitrified melanosomes. This made it possible to probe structural features with a nanofocus locally, and hence shed light on surface versus bulk features separately.

Fields of vitrified B6 and D2 melanosome samples were imaged by optical microscopy and STXM, under cryogenic conditions (Fig. 2). An area of each sample was scanned with the X-ray beam, and a darkfield STXM image was produced by integrating the scattered photons in each raster point, plotting the intensities pixel-wise according to the beam position on the samples, and correlating optical and X-ray data to identify single melanosomes.

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Figure 2. Correlation of maps generated by optical phase-contrast microscopy, cryo microscopy and darkfield cryo-STXM.

Melanosomes of the genotypes C57BL/6J (B6) and DBA/2J (D2) are shown in tiles (A–C) and (D–F), respectively. In both cases, the sample is embedded in an amorphous ice matrix. The optical micrographs, recorded at 100 K, are superimposed with a semi-transparent (B and E) and an opaque (C and F) STXM map. Note that despite the fact that melanosome density is comparable in the two samples, the STXM images feature significant differences in signal intensity as well as the spatial distribution of that signal, revealing structural differences between the melanosomes of the two genotypes. The area of the scanned regions is 20.4×20.4 µm2.

https://doi.org/10.1371/journal.pone.0090884.g002

This analysis revealed qualitative differences between B6 and D2 melanosomes. Even though optical micrographs suggested that the two were of similar melanosome density (Fig. 2), the STXM maps of B6 melanosomes revealed a sparse scattering intensity compared to those of the D2 organelles. This effect is illustrated quantitatively in the plot presented in Fig. 3A. The graph consists of two histograms that represent the intensity distributions within the darkfield STXM maps shown in Fig. 2, with the intensities normalized to the maximum value in the D2 scan. Notably, the intensity of the signal in the B6 darkfield STXM map is lower than that in the D2 map. Moreover, the distributions of intensity in the two samples are of significantly different width. Whereas the distribution is rather broad in the case of the D2 organelles, it is almost binary in the case of the B6 melanosomes, with scatter at a particular signal intensity either detectable or not.

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Figure 3. X-ray scattering data for cryogenically prepared melanosomes from C57BL/6J (B6) and DBA/2J (D2) mice.

(A) Normalized intensity histograms derived from darkfield STXM maps of B6 and D2 samples like those shown in Fig. 2. The intensity of scatter for the D2 melanosomes is higher and has a broader distribution, than that for the B6 sample. The photon energy was 7.9 keV. (B) Sum of 13 background-corrected scattering events, i.e. hits, for a B6 sample. The scattering pattern is anisotropic. (C) Sum of 13 background-corrected scattering events for a D2 sample. The scattering pattern is isotropic. In (B) and (C), intensity is color-coded on a logarithmic scale. The horizontal bars in (B) and (C), which are devoid of signal, correspond to insensitive regions of the PILATUS, and separate the three detector modules. The artificial look of the regions in the centers is due to the stacking of two semi-transparent beamstops.

https://doi.org/10.1371/journal.pone.0090884.g003

Analysis of 13 accumulated, background-corrected scattering events (Fig. 3B and 3C) of individual melanosomes from each strain indicated that B6 melanosomes exhibit anisotropic scattering patterns whereas D2 melanosomes have comparatively isotropic scattering. Because hard X-rays are far more influenced by three-dimensional structures and granularities on the nanoscale than the visible wavelengths used in optical microscopy, the observation of different scattering characteristics (binary vs. broad and directed vs. isotropic) again indicates that the B6 and D2 melanosomes have different levels of overall complexity.