Quantitative chemical analysis of ocular melanosomes in stained and non-stained tissues

Energy-filtered Analytical Electron Microscopy (AEM) was used to image the ultrastructure and determine quantitatively the chemical composition of rat melanosomes of the choroid and the Retinal Pigment Epithelium (RPE). For the first time, the effect of staining in elemental analysis of melanosomes was investigated. Detection limits and accuracies of the applied methods were determined. Compared to previous work applying only quantitative Energy Dispersive X-ray microanalysis (EDX) in the TEM (Eibl, O., et al., 2006. Micron 37, 262), here we present a combined quantitative EDX and Electron Energy Loss Spectroscopy (EELS) analysis, including N. This yields the fraction of eumelanin and pheomelanin in melanosomes by the S/N mole fraction ratio. Melanosomes of the sepia ink sac, used as eumelanin standard, showed an S/N mole fraction ratio of <0.004. Thus, they consist primarily of eumelanin as reported by degradation analysis. In contrast, melanosomes of the rats contained mixed melanin with significant amounts of pheomelanin (S/N 0.02) in the RPE and the choroid. Consistent with the previous publication, it was shown that oxygen mole fractions are especially large in melanosomes (7-10 at.%) compared to other cell compartments, e.g. 2-4 at.% oxygen in the cytoplasm. In the melanosomes of non-stained tissue, the oxygen mole fraction clearly correlated with the Ca mole fraction. EDX spectra used for quantitative analysis had about 15,000 net counts under the oxygen peak, which is necessary to obtain (i) a small statistical error for oxygen and (ii) optimum minimum detectable mole fractions for S, Ca and transition metals. The precise determination of the oxygen mole fraction in melanosomes is important for understanding metabolism. Therefore, a detailed analysis was carried out on the possible errors affecting quantification. While O, S, and N mole fractions yielded similar results in stained and non-stained ocular melanosomes of rats, transition metals can only be determined reliably in non-stained tissues. High-precision EDX analysis of melanosomes yielded minimum detectable mole fractions of less than 0.04 at.% for Cu and Zn, these elements were present in melanosomes with mole fractions of about 0.3 at.% and 0.1at.%, respectively. Zn is of great importance for metabolism and for age related macular degeneration. Its mole fraction in melanosomes of rats is large enough to be detected and to be quantitatively analyzed by EDX spectroscopy. Ultrastructural information can now be correlated to the elemental composition. This is important to better understand the physical and chemical properties of melanosomal metabolism and turnover.