Over the last year, we have explored the potential of this sequential slice-and-view strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue. We first established that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also showed that 200 nm thick sections can be generated in situ by milling of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. In an extension of these studies we have shown 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 show 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. 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 are using ion-abrasion scanning electron microscopy to provide rapid feedback on subcellular localization of these nanoparticles in an effort that could be highly 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.
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