Imaging cellular assemblies with three-dimensional electron microscopy

Division Of Basic Sciences - Nci

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Abstract

Over the last year, we used ion abrasion scanning electron microscopy (IA-SEM) to show that MNT-1 melanoma cells can be rapidly imaged at resolutions of 30-nm in the z-direction (direction of section removal), and 6 nm in the x-y plane (plane of section removal). We also showed that individual gold and quantum dot particles can be localized in the images, demonstrating that ion-abrasion scanning electron microscopy is a powerful method for obtaining combined information on 3D ultrastructure and molecular localization. In particular, statistical analysis of information obtained from the imaging such as size, shape and compositional analysis of organelles could provide valuable diagnostic markers for discriminating normal cells from abnormal cells. Further, we reported the first application of IA-SEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms, sometimes referred to as Natures nanofactory 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. Yet another aspect of these studies concerns applications for clinical and pre-clinical imaging of tissue specimens. Nanoparticles such as gold and iron oxide-based compounds that have electron dense features are especially amenable to detection as imaging agents. We have used ion-abrasion scanning electron microscopy to determine subcellular localization of these nanoparticles in an effort that could be relevant in clinical contexts to determine useful doses, efficiency of tissue targeting, and efficacy of drug delivery to the correct targets. Knowledge of drug distribution could also lead to ideas for chemical modifications that could improve delivery of these nanoscale reagents. In tomographic studies of the predatory bacterium Bdellovibrio bacteriovorus, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformations of their outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 minutes, and analysis of the internal architecture of highly bent cells shows that the overall distribution of molecular machines and the nucleoid is similar to those seen in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We have suggested that rearrangements of these structures, in combination with the unique properties of the cell envelope may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey. We have used electron tomography to analyze structures of nanoparticles such as the icosahedral pyruvate dehydrogenase (PDH) enzyme complex and the cancer drug Doxil. We have shown that electron tomography can be a powerful method for providing quality control on the physical characteristics of complex nanomedicine formulations such as Doxil. We have also shown that individual PDH complexes can be imaged and interpreted in terms of the atomic structures of the E1, E2 and E3 components, further establishing an important role for tomography as a structural tool in nanomedicine.