We have studied the iron metabolism in nine patients with erythropoietic protoporphyria (EPP) and three patients with sideroblastic anaemia (SA). All, except one EPP patient were iron deficient. The SA patients had a secondary haemochromatosis. The bone marrow aspirates of patients with SA and also three patients with EPP had a high incidence of ring sideroblasts. Ultrastructural examination of the bone marrow consistently showed finely dispersed electron-dense deposits localized in mitochondria of erythroblasts in all patients with EPP and SA. Mitochondrial electron energy-loss spectroscopy (EELS) indicated identical iron compounds in erythroblasts of all EPP and SA patients. These findings indicate that the mitochondrial iron utilization is disturbed in EPP and SA. The observation of mitochondrial iron deposition in erythroblasts in EPP and SA suggests that this failure is not of pathognomonic value for diagnosis of SA, but is apparently the result of an inefficient haem synthesis, in EPP due to a defective ferrochelatase. The mitochondrial iron deposition does not depend on the iron status (iron overload or iron deficiency) of the EPP patient.

SUMMARY Electron energy‐loss spectroscopy (EELS) has been used to determine elemental concentrations in biological specimens, consisting of ultrathin‐sectioned cells and tissues. Chelex100‐based Ca‐ and Fe Bio‐standards are used for elemental quantification to establish iron and calcium concentrations. These Bio‐standards, as well as the biological materials, are treated in a standard EM procedure such that ‘known’ and ‘unknown’ sites are located in one cross‐section. Uncertainties and variabilities present in the equations for calculating the concentration in the ‘unknown’ site (determined by comparing simplex‐fitted EEL spectra from Bio‐standards with those from tissue) are outlined in two examples. Using an H+ Bio‐standard, the matrix composition of such biological cell material is analysed, leading to values which approach each other closely. Quantitative EELS, using Chelex100‐based Bio‐standards, is advocated.

SUMMARY A computer program for quantitative spectral analysis is proposed for the elemental analysis of biological material by electron energy‐loss spectroscopy in a conventional transmission electron microscope, the Zeiss EM902. Bio‐standards are used to test the performance of this program. The application of a simplex optimization method for curve‐fitting is proposed to separate the ionization edge from the background. Making use of Ce‐, Ca‐ and Fe‐bio‐standards, this method is compared with Egerton's well‐known two‐area method.

Microwave irradiation has been applied to reduce the immunogold staining time of ultrathin sections of Lowicryl embedded specimens. Labelling has been stimulated by microwave irradiation during incubation with 10 nm colloidal gold particles coated with either goat anti-mouse antibodies (GaM-gold) or goat anti-rabbit antibodies (GaR-gold) and has been compared with control incubations. Quantification has been performed on cytoplasmic membranes or lysosomes labelled with a primary antibody. Counting the gold particles over specific and non-specific sites in electron micrographs and electron microscopic images by IBAS 2000 revealed that irradiation of 25 microliters droplets both at 80 W and 150 W resulted in an accelerated immunogold labelling, while the non-specific background levels were not increased. A plateau level in immunogold labelling intensity was reached after 25 min incubation under microwave irradiation at 150 W as compared to 120 min incubation without microwaves. No improvement in localization sharpness of immunogold labelling on membranes was achieved by microwave irradiation. The microwave-mediated acceleration of immunogold staining may be considered as an example of a staining method with a restricted thermal action on microvolumes as indicated by direct temperature measurements using a fibre-optic thermometer.

We have used quantitative immunoelectronmicroscopy to compare the in situ localization of acid alpha-glucosidase, lysosomal acid phosphatase, beta-hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid alpha-glucosidase than it is for beta-hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.