Imaging cellular assemblies with three-dimensional electron microscopy

在过去的一年里，我们已经探索了这种连续切片和视图策略的潜力，用于冷冻酵母细胞和肿瘤组织的特定位点3D成像。我们首先确定了这种方法可以识别细胞内特征的位置，例如100纳米宽的酵母核孔复合物。我们还发现，通过使用离子束对树脂包埋的标本进行铣削，可以在原位生成200 nm厚的切片，这为使用超微切片机对细胞和组织进行人工切片提供了一种有价值的替代方法。在这些研究的扩展中，我们已经证明MNT-1黑色素瘤细胞可以在z方向（切片去除方向）以30 nm的分辨率快速成像，在x-y平面（切片去除平面）以6 nm的分辨率快速成像。我们还表明，单个金粒子和量子点粒子可以在图像中定位，这表明离子磨损扫描电子显微镜是获得三维超微结构和分子定位综合信息的有力方法。特别是对成像所得信息的统计分析，如细胞器的大小、形状和成分分析，可以为区分正常细胞和异常细胞提供有价值的诊断标记。然而，这些研究的另一个方面涉及组织标本临床和临床前成像的应用。纳米粒子，如氧化金和氧化铁化合物，具有电子密度特征，特别适合作为显像剂检测。我们正在使用离子磨损扫描电子显微镜对这些纳米颗粒的亚细胞定位提供快速反馈，这可能与临床环境高度相关，以确定有用剂量、组织靶向效率和药物递送到正确靶点的功效。对药物分布的了解也可能导致化学修饰的想法，从而改善这些纳米级试剂的输送。

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.