Scanning Electron Microscopy


Morphometrical and X-ray elemental information was extracted from Scanning Transmission Electron Microscopy (STEM) images of hepatic lysosomes of a patient with idiopathic hemosiderosis before and after treatment by phlebotomy. The elements of interest were (i) iron, stored in pathological quantities in hepatic lysosomal structures and (ii) cerium, used as a capture ion after a cytochemical reaction to detect acid phosphatase activity in the lysosomal structures.

Morphologically the lysosomal structures are heteromorph and the elements iron and cerium are heterogeneously distributed. With "reduced raster" (= reduced scanning area) analysis at 16 x 16 pixel points (integrating image and X-ray microanalysis), a marked difference in the area of the cross sectioned lysosomal structures before and after treatment could be demonstrated. Simultaneously the difference in the relative orientation of the elements iron and cerium before and after phlebotomy could be visualized.

Chelex ion exchange beads, loaded with 11.5% w/w iron, and coembedded with the tissue blocks, were used as an internal standard. A mean iron peak to background ratio was obtained and a factor, converting ratio to absolute iron concentration, was calculated. The same calculation procedure, now per pixel point, was followed for the hepatic lysosomal structures. A marked difference in iron concentration in the individual lysosomal structures was observed before and after treatment by phlebotomy.

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