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Antje K. Biesemeier, Oliver Eibl, Ulrich Schraermeyer; Melanosomes And Lipofuscin Granules Of The RPE Can Be Distinguished By Quantitative Analytical Electron Microscopy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):905.
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© ARVO (1962-2015); The Authors (2016-present)
To distinguish different pigment granule types of the retinal pigment epithelium (RPE) by their chemical composition with high accuracy analytical electron microscopy (AEM). This can be achieved without prior isolation of the pigments from the tissue and with a lateral resolution of 10 nm.
The eyes of two healthy human donors (68 and 85 years) were embedded in Epon. Energy-filtered TEM and combined quantitative EDX and EELS analysis were applied to analyze the chemical composition of RPE pigment granules, yielding minimum detectable mole fractions of about 0.02 at% for Cu and Zn by EDX.
Six melanosomes (M), melanolipofuscin (ML) and lipofuscin (L) granules per eye were investigated. Mean mole fractions of the different pigments are summarized in Table1. They show precisely the different composition of melanosomes and lipofuscin concerning N, O, P, S, Cu and Zn mole fractions. The S/N ratio of 0.18 yielded high pheomelanin content for the melanosomes. The dark fraction of ML core-shell structures showed a chemical composition close to the melanosomes and the composition of the bright fraction was close to that of lipofuscin, despite a higher N in bright ML. Differences between the donors, which are responsible for high standard deviations in O and Ca mole fractions in this work, will be investigated on a larger set of samples.
By EDX and EELS analysis, the composition of melanosomes and lipofuscin granules can now clearly be distinguished in electron micrographs. We suggest a lack of proteins in lipofuscin due to negligible N mole fractions and a lack of phospholipids in melanosomes due to negligible P mole fractions, respectively. Trace elements like Zn are of great importance for metabolism and anti-oxidative mechanisms in melanosomes and can also be investigated by these methods. Therefore, chemical microanalysis of RPE pigment granules yields new perspectives for understanding age-related and disease-related changes in elemental composition.
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