The Microstructural Reconstruction of Variously Sintered Ni-SDC Cermets Using Focused Ion Beam Scanning Electron Microscopy Nanotomography.

This work focuses in-depth on the quantitative relationships between primary first-order microstructural parameters (i.e., volume fractions of various phases and particle size distribution) with the more complex second-order topological features (i.e., connectivity of phases, three-phase boundary length (TPBL), interfacial areas, or tortuosity). As a suitable model material, a cermet nickel/samaria-doped ceria (Ni-SDC) is used as an anode in a solid oxide fuel cell (SOFC). A microstructure description of nano-sized Ni-SDC cermets, fabricated at various sintering conditions from 1100 °C to 1400 °C, was performed using FIB-SEM nanotomography. The samples were serially sectioned employing a fully automated slicing procedure with active drift correction algorithms and an auto-focusing routine to obtain a series of low-loss BSE images. Advanced image processing algorithms were developed and applied directly to image data volume. The microstructural-topological relationships are crucial for the microstructure optimisation and, thus, the improvement of the corresponding electrode performance. Since all grains of individual phases (Ni, SDC, or pores) did not percolate, special attention was given to the visualisation of the so-called active TPBL. Based on the determined microstructure characteristics of the prepared Ni-SDC cermets, including simulations of gas flow and pressure drop, thermal treatment at 1200 °C was recognised as the most appropriate sintering temperature.

Benefits and Limitations of Low-kV Macromolecular Imaging of Frozen-Hydrated Biological Samples.

Object contrast is one of the most important parameters of macromolecular imaging. Low-voltage transmission electron microscopy has shown an increased atom contrast for carbon materials, indicating that amplitude contrast contributions increase at a higher rate than phase contrast and inelastic scattering. Here, we studied image contrast using ice-embedded tobacco mosaic virus particles as test samples at 20-80 keV electron energy. The particles showed the expected increase in contrast for lower energies, but at the same time the 2.3-nm-resolution measure decayed more rapidly. We found a pronounced signal loss below 60 keV, and therefore we conclude that increased inelastic scattering counteracts increased amplitude contrast. This model also implies that as long as the amplitude contrast does not increase with resolution, beam damage and multiple scattering will always win over increased contrast at the lowest energies. Therefore, we cannot expect that low-energy imaging of conventionally prepared samples would provide better data than state-of-the-art 200-300 keV imaging.

Tomographic reconstruction reveals the morphology of a unique cellular organelle, the aggregated macrotubules (Macrotubuli aggregati) of human retinal horizontal cells.

Horizontal cells of the human retina contain unique tubular organelles that have a diameter which is about 10 times larger than that of microtubules (~230 nm). These macrotubuli in most cases form regular aggregates. Therefore we propose to introduce them as Macrotubuli aggregati in the Terminologia histologica. Tomographic investigation of the structures revealed that the walls of the tubules most probably consist of intermediate filaments running nearly parallel to each other and show somewhat regularly attached ribosomes on their inner and also outer surface. About 2% of the organelles exhibit double- to multiple layered walls and less than 1% resemble large scrolls. The tubules may extend 10 to over 20 µm in the cytoplasm and are also encountered in soma-near processes extending into the outer plexiform layer. It remains unclear why these structures are only present in humans and few other species and why almost only in horizontal cells. Speculations on possible functions are discussed.

The immune control of HTLV-1 infection: selection forces and dynamics.

Cytotoxic T lymphocytes (CTLs) play a central role in the protective immune response to human T-lymphotropic virus 1 (HTLV-1). Here we consider two questions. First, what determines the strength of an individual's HTLV-1-specific CTL response? Second, what controls the rate of expression of HTLV-1 in vivo, which is greater in patients with HAM/TSP than in asymptomatic carriers with the same proviral load? Recent evidence shows that FoxP3+CD4+ T cells are abnormally frequent in HTLV-1 infection, and the frequency of these cells is inversely correlated with the rate of CTL lysis of HTLV-1-infected cells, suggesting that FoxP3+CD4+ cell frequency is an important determinant of the outcome of HTLV-1 infection. There is also new evidence that the rate of expression of HTLV-1 in vivo is associated with the transcriptional activity of the flanking host genome. We suggest that the frequencies of HTLV-1-infected T cell clones in vivo are determined by a dynamic balance between positive and negative selection forces that differ among the clones.

Human T-lymphotropic virus-1 visualized at the virological synapse by electron tomography.

Human T-lymphotropic virus 1 (HTLV-1) is transmitted directly between cells via an organized cell-cell contact called a virological synapse (VS). The VS has been studied by light microscopy, but the ultrastructure of the VS and the nature of the transmitted viral particle have remained unknown. Cell-free enveloped virions of HTLV-1 are undetectable in the serum of individuals infected with the human T-lymphotropic virus 1 (HTLV-1) and during in vitro culture of naturally infected lymphocytes. However, the viral envelope protein is required for infectivity of HTLV-1, suggesting that complete, enveloped HTLV-1 virions are transferred across the synapse. Here, we use electron tomography combined with immunostaining of viral protein to demonstrate the presence of enveloped HTLV-1 particles within the VS formed between naturally infected lymphocytes. We show in 3D that HTLV-1 particles can be detected in multiple synaptic clefts at different locations simultaneously within the same VS. The synaptic clefts are surrounded by the tightly apposed plasma membranes of the two cells. HTLV-1 virions can contact the recipient cell membrane before detaching from the infected cell. The results show that the HTLV-1 virological synapse that forms spontaneously between lymphocytes of HTLV-1 infected individuals allows direct cell-cell transmission of the virus by triggered, directional release of enveloped HTLV-1 particles into confined intercellular spaces.

Centrosome polarization delivers secretory granules to the immunological synapse.

Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.