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.

Chitin‐Based Organic Networks: An Integral Part of Cell Wall Biosilica in the Diatom Thalassiosira pseudonana

“…[9] Recent ion-abrasion scanning electron microscopic [10] as well as atomic force microscopic studies [11] on cell wall formation in T. pseudonana revealed the presence of filamentous nano-and microscale structures within the growing cell wall which apparently contain central templating organic structures ("linear proteins"). [10] Chitin (poly-N-acetyl-d-glucosamine) occurs in numerous calciumbased biominerals. [12] It is assumed to form insoluble scaffolds or compartments, wherein chitin-associated biomolecules control calcium biomineralization events.…”

Three-dimensional imaging of human stem cells using soft X-ray tomography

“…Although hard X-ray tomography instruments which achieve sub-100 nm resolution exist, biological specimens have very low absorption in the hard X-ray region, imparting challenges in using this technique for their analysis. One approach to bypass this limitation involves 200-500 nm ultramicrotome sectioning for the visualization of intracellular components [17]; however, this laborious process has prevented widespread application of the technique. A number of researchers are pursuing phase imaging to overcome this limitation [18][19][20] and it will be interesting to track the development of the hard X-ray tomography technique and see if it can successfully be applied for cellular imaging at resolutions relevant to resolve organelles.…”

Effectiveness of Cleaning Methods for Investigating Diatoms

“…Chemical cleaning can destroy some features of diatoms useful for taxonomy or other studies, especially those diatoms constructed of thin or delicate silicious-organic frustules, processes or with chitin strands. Researchers wanting to study 3D frustule structure [4], the organic casing [6], energy dispersive x-ray spectroscopy (EDS) and structures of mineral-organics [4], polysaccharide and chitin secretions, structural and connective relationships with epiphytic, symbiotic or parasitic organisms, such as bacteria or diatoms [6], may require other techniques for gentle and controlled cleaning. Thus we researched different chemical methods and plasma, UV and FIB instruments to determine their effectiveness in a wide range of applications that require gentle, staged and controlled removal of organics or cutting and cross-sectioning of diatom cell structures.…”