3D Imaging of Diatoms with Ion Abrasion Scanning Electron Microscopy

Abstract

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 ~ 30 nm 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. 3

Compartmental Analysis Suggests Macropinocytosis at the Onset of Diatom Valve Formation

“…The crux is that in macropinocytosis one larger vesicle is formed, which is in line with recent observations on the diatom Thalassiosira pseudonana [16] and observed presence of vacuolar-like structures with different functions [6].…”

“…It remains to be shown whether or not this process takes place by: 1) 'classical' pinocytosis in which multiple smaller vesicles are involved, or 2) macropinocytosis in which one larger vesicle is formed which transforms to become the initial SDV. The macropinocytosis hypothesis is supported by recent 3-D ultrastructural studies on SDV transformations [16] in which actin filaments are specifically involved in molding and/or compressing the SDV at the cleavage furrow [17]; SDV transformation agrees well with an earlier observed high membrane activity in dividing diatom cells [22,23]. Furthermore, in plants a similar uptake mechanism for silicon has been observed [24].…”

“…Ionic strength determines the degree of condensation of the silica and affects the nanostructure of the solid silica formed in diatoms [7]. Noteworthy is that in diatoms early valve formation coincides with nanostructuring of the primer two-dimensional silica base [16,17]. The impact of reaction conditions on silica polymerization in diatom frustule formation suggests that cellular transport routes during which polymerization of accumulated silicon species could occur are not likely to be involved in valve formation [4,7].…”

3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells

“…approach for 3D structural analysis of whole cells at a resolution of ∼20 nm (16,17,20,21). With this imaging strategy, cells or tissues are exposed to a scanning gallium ion beam that iteratively abrades the specimen surface to remove a layer of defined thickness (typically ∼15-20 nm) for each pass.…”

“…Although these and similar experiments with other kinds of cell-cell synapses (14,15) have provided informative glimpses of the nature of cell-cell contact, imaging experiments using conventional 2D TEM provide only crosssectional views. The focus of the experiments presented here is to describe the 3D architecture of virological synapses using two emerging technologies for 3D electron microscopy: ion abrasion (IA) SEM (16,17) and electron tomography (18). Our experiments use these imaging approaches to reveal the 3D arrangement of virions and virion-containing compartments at the earliest stages of contact between HIV-pulsed mature dendritic cells and uninfected T cells when virological synapses have been established but before productive infection of either the dendritic cell or the T cell has occurred.…”