Ion-abrasion Scanning Electron Microscopy Research Articles

Three-Dimensional Imaging of Cerebellar Mossy Fiber Rosettes by Ion-Abrasion Scanning Electron Microscopy

The detailed knowledge of the three-dimensional (3D) organization of the nervous tissue provides essential information on its functional elucidation. We used serial block-face scanning electron microscopy with focused ion beam (FIB) milling to reveal 3D morphologies of the mossy fiber rosettes in the mice cerebellum. Three-week-old C57 black mice were perfused with a fixative of 1% paraformaldehyde/1% glutaraldehyde in phosphate buffer; the cerebellum was osmicated and embedded in the Araldite. The block containing granule cell layer was sliced with FIB and observed by field-emission scanning electron microscopy. The contrast of backscattered electron image of the block-face was similar to that of transmission electron microscopy and processed using 3D visualization software for further analysis. The mossy fiber rosettes on each image were segmented and rendered to visualize the 3D model. The complete 3D characters of the mossy fiber rosette could be browsed on the A-Works, in-house software, and some preliminary quantitative data on synapse of the rosette could be extracted from these models. Thanks to the development of two-beam imaging and optimized software, we could get 3D information on cerebellar mossy fiber rosettes with ease and speedily, which would be an additive choice to explore 3D structures of the nervous systems and their networks.

Visualizing cells and humans in 3D: biomedical image analysis at nanometer and meter scales.

Researchers analyzed and presented volume data from the Visible Human Project (VHP) and data from high-resolution 3D ion-abrasion scanning electron microscopy (IA-SEM). They acquired the VHP data using cryosectioning, a destructive approach to 3D human anatomical imaging resulting in whole-body images with a field of view approaching 2 meters and a minimum resolvable feature size of 300 microns. IA-SEM is a type of block-face imaging microscopy, a destructive approach to microscopic 3D imaging of cells. The field of view of IA-SEM data is on the order of 10 microns (whole cell) with a minimum resolvable feature size of 15 nanometers (single-slice thickness). Despite the difference in subject and scale, the analysis and modeling methods were remarkably similar. They are derived from image processing, computer vision, and computer graphics techniques. Moreover, together we are employing medical illustration, visualization, and rapid prototyping to inform and inspire biomedical science. By combining graphics and biology, we are imaging across nine orders of magnitude of space to better promote public health through research.

HIV-1 activates Cdc42 and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation

AbstractHIV-1 cell-to-cell transmission confers a strong advantage as it increases efficiency of transfer up to 100-fold compared with a cell-free route. Mechanisms of HIV-1 cell-to-cell transmission are still unclear and can in part be explained by the presence of actin-containing cellular protrusions. Such protrusions have been shown to facilitate cell-to-cell viral dissemination. Using fluorescence microscopy, electron tomography, and ion abrasion scanning electron microscopy we show that HIV-1 induces membrane extensions in immature dendritic cells through activation of Cdc42. We demonstrate that these extensions are induced after engagement of DC-SIGN by HIV-1env via a cascade that involves Src kinases, Cdc42, Pak1, and Wasp. Silencing of Cdc42 or treatment with a specific Cdc42 inhibitor, Secramine A, dramatically reduced the number of membrane protrusions visualized on the cell surface and decreased HIV-1 transfer via infectious synapses. Ion abrasion scanning electron microscopy of cell-cell contact regions showed that cellular extensions from immature dendritic cells that have the appearance of thin filopodia in thin section images are indeed extended membranous sheets with a narrow cross section. Our results demonstrate that HIV-1 binding on immature dendritic cells enhances the formation of membrane extensions that facilitate HIV-1 transfer to CD4+ T lymphocytes.

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

3D Imaging of Dendritic Cells Using Serial Ion-Abrasion Scanning Electron Microscopy (SIASEM) Reveals That HIV-1 is Stored Within Surface-connected Membrane Folds

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Ion-abrasion scanning electron microscopy reveals distorted liver mitochondrial morphology in murine methylmalonic acidemia

Methylmalonic acidemia is a lethal inborn error of metabolism that causes mitochondrial impairment, multi-organ dysfunction and a shortened lifespan. Previous transmission electron microscope studies of thin sections from normal (Mut(+/+)) and diseased (Mut(-/-)) tissue found that the mitochondria appear to occupy a progressively larger volume of mutant cells with age, becoming megamitochondria. To assess changes in shape and volume of mitochondria resulting from the mutation, we carried out ion-abrasion scanning electron microscopy (IA-SEM), a method for 3D imaging that involves the iterative use of a focused gallium ion beam to abrade the surface of the specimen, and a scanning electron beam to image the newly exposed surface. Using IA-SEM, we show that mitochondria are more convoluted and have a broader distribution of sizes in the mutant tissue. Compared to normal cells, mitochondria from mutant cells have a larger surface-area-to-volume ratio, which can be attributed to their convoluted shape and not to their elongation or reduced volume. The 3D imaging approach and image analysis described here could therefore be useful as a diagnostic tool for the evaluation of disease progression in aberrant cells at resolutions higher than that currently achieved using confocal light microscopy.

Ion-Abrasion Scanning Electron Microscopy Reveals Surface-Connected Tubular Conduits in HIV-Infected Macrophages

HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity, and 3D distribution of these compartments has remained controversial. Here, we report the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM, we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of ∼150–200 nm, and lengths of up to ∼5 µm that extend to the cell surface from vesicular compartments that contain assembling HIV-1 virions. These types of surface-connected tubular compartments are not observed in T cells infected with the 29/31 KE Gag-matrix mutant where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation.

3D imaging of diatoms with ion-abrasion scanning electron microscopy

Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at approximately 30nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the "hard" (siliceous) and "soft" (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system.

3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy

Understanding the hierarchical organization of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. We are using ion-abrasion scanning electron microscopy (IA-SEM) to visualize this hierarchical organization in an approach that combines focused ion-beam milling with scanning electron microscopy. Here, we extend our previous studies on imaging yeast cells to image subcellular architecture in human melanoma cells and melanocytes at resolutions as high as approximately 6 and approximately 20 nm in the directions parallel and perpendicular, respectively, to the direction of ion-beam milling. The 3D images demonstrate the striking spatial relationships between specific organelles such as mitochondria and membranes of the endoplasmic reticulum, and the distribution of unique cellular components such as melanosomes. We also show that 10nm-sized gold particles and quantum dot particles with 7 nm-sized cores can be detected in single cross-sectional images. IA-SEM is thus a useful tool for imaging large mammalian cells in their entirety at resolutions in the nanometer range.

3D Imaging of Diatoms with Ion Abrasion Scanning Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008