Aims/hypothesis IA-2 and IA-2β are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of these genes impairs the secretion of insulin by reducing the number of DCV. Methods Insulin secretion, content and DCV number were evaluated in islets from single knockout (IA-2 KO, IA-2β KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca2+ currents, DCV half-life, lysosome number and size and autophagy. Results Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (P < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca2+ currents support these findings. Electron microscopy of islets from KO mice revealed a marked increase (P < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (P < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO as compared to WT mice (P < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV. Conclusions/interpretation We conclude that the decrease in insulin content and secretion, resulting from the deletion of IA-2 and/or IA-2β, is due to a decrease in the number of DCV.
A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 atomic % osmium in a carbon matrix, it was found that for 1-μm thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF-STEM tomographic tilt series from a stained 600-nm thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF-STEM but significantly less in the BF-STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF-STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.
We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9 ± 5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections.
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