Technology Development for 3D Electron Microscopy

我们在过去几年的工作中开发了许多新技术，这些技术现在是我们在艾滋病毒/艾滋病和癌症生物学领域发现工作的核心。现将进展特别值得注意的两个领域总结如下。在过去的一年中，我们实验室3D成像技术基础设施的发展使我们能够分析许多HIV-1/SIV刺突和刺突复杂结构。这是前所未有的，因为在2008年，我们通过确定前三个HIV-1刺突复合体结构取得了突破。我们精简的实验和计算程序使我们能够全天候远程操作，平均仪器正常运行时间为85%，用于数据收集。在接下来的几年里，我们将继续完善这条管道，增加使用内联、无监督分类程序分离不同构象的工具。一个被证明是有用的进展是数据处理策略的发展，允许明确分离封闭和开放状态，以及三聚体Env的非配体和抗体配体状态，当它们存在于混合物中时。我们已经证明，识别和去除具有最低信噪比的峰值可以提高单个峰值之间校准的总体准确性，而校准精度反过来又决定了在异质SIV和HIV-1混合物中评估Env构象异质性的图像分类的成功。我们通过成功分离和重建同时存在于混合物中的非配体和抗体配体HIV-1 Env复合物的不同3D结构，验证了这些计算分离程序。了解细胞内蛋白质、代谢物和元素的空间分布对了解健康和疾病的功能具有潜在的重要意义。定位感兴趣的亚细胞区域需要亚微米分辨率，基于光学技术的技术，如荧光显微镜，已被证明对监测细胞内部和周围的离散化学变化非常有用。然而，荧光成像仅限于成像天然荧光分子或那些已经特别附属于感兴趣的分析物。相比之下，基于质谱的技术为实现同时检测多种未标记的细胞成分提供了独特的机会。基质辅助激光解吸电离质谱（MALDI-MS）已广泛用于组织成像，尽管典型的空间分辨率（约20至50微米）使其不适合大多数哺乳动物细胞的亚细胞成像。基于次级离子质谱（SIMS）的方法，SIMS的主离子束小至50 nm至500 nm，具有更高的分辨率，因此对单细胞化学成像质谱法很有用。尽管取得了这些进步，SIMS成像仍然很少有生物学发现，而且在亚细胞分子成像方面也只取得了一定的成功，因为它有几个缺点，比如与MALDI-MS相比质量范围更小（SIMS的质量范围通常小于500 m/z），初级光束诱导的分子损伤水平更高，目标分子的分子电离概率通常较低。此外，在开发维持细胞化学完整性的样品制备方案、最大限度地减少入射光束对分子的损伤（这限制了生成3D数据集的能力）以及实现更好的空间分辨率的理论极限方面也存在挑战。通过将SIMS与聚焦离子束（FIB）铣削相结合，我们在样品制备和将成像扩展到第三维度的策略方面都取得了进展。利用这种方法，我们利用二次离子质谱法对完整哺乳动物细胞内的化学物质进行了成像，同时对亚细胞元素和分子物种以及固有的膜特异性细胞标志物进行了制图。通过特定位置聚焦离子束铣削暴露的细胞表面和细胞内部成像结果表明，可以实现约400 nm的面内分辨率。绘制细胞表面磷脂酰胆碱和细胞中存在的其他几个分子离子的结果表明，单个细胞的空间分辨化学特征可以通过对不同m/z比获得的分子图像进行新颖的多变量分析和分类得到。我们建立的标本制备和细胞内部化学成像方法为获得未染色哺乳动物细胞的3D分子图谱提供了基础，特别是与探测小分子（如药物和代谢物）的亚细胞分布有关。

A number of new technologies have been developed from our work over the last few years that are now at the core of our discovery efforts in the areas of HIV/AIDS and cancer biology. Two areas where progress that been especially noteworthy are summarized below. The development of the technological infrastructure for 3D imaging in our laboratory has enabled the analysis, over the last year, of numerous HIV-1/SIV spike and spike complex structures. This is unprecedented, given that in 2008, we achieved a breakthrough by determining the first three HIV-1 spike complex structures. Our streamlined experimental and computational procedures allow us to operate round the clock, remotely, and with average instrument uptimes of 85% for data collection. Over the coming years, we will continue to refine this pipeline by adding tools for separating distinct conformations using in-line, unsupervised classification procedures. One advance that is proving to be useful is the development of data processing strategies that allow clear separation of the closed and open states, as well as unliganded and antibody liganded states of trimeric Env when they are present in mixtures. We have shown that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual spikes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity of Env in heterogeneous SIV and HIV-1 mixtures. We have validate these procedures for computational separation by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded HIV-1 Env complexes simultaneously present in a mixture. Knowledge of the spatial distributions of proteins, metabolites and elements within the cell is potentially important for understanding function in health and disease. Submicrometer resolution is required for localizing subcellular regions of interest, and techniques based on optical technology, such as fluorescence microscopy, have proven to be very useful for monitoring discrete chemical changes within and around cells. However, fluorescence imaging is restricted to imaging natively fluorescent molecules or those that have been specifically attached to analytes of interest. In contrast, mass spectrometry-based techniques provide unique opportunities for achieving simultaneous detection of multiple, unlabeled cellular components. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used extensively for tissue imaging, although the typical spatial resolutions achieved (around 20 to 50 microns) make it unsuitable for subcellular imaging of most mammalian cells. Secondary ion mass spectrometry (SIMS)-based approaches, with SIMS primary ion beams as small as 50 nm to 500 nm, are capable of much higher resolution, and are therefore useful for single cell chemical imaging mass spectrometry. Despite these advances, SIMS imaging continues to yield few biological discoveries and only modest success with subcellular molecular imaging because of several shortcomings such as smaller mass ranges compared to MALDI-MS (typically less than 500 m/z for SIMS), higher primary beam-induced molecular damage levels, and generally low molecular ionization probabilities of target molecules. Further, there have also been challenges in developing sample preparation protocols that maintain the chemical integrity of the cells, in minimizing molecular damage from the incident beam that limits the ability to generate 3D data sets, and achieving the theoretical limits of better spatial resolution. We have made advances both in specimen preparation and in strategies to extend the imaging into the third dimension by combining SIMS with focused ion beam (FIB) milling. Using this approach we have imaged chemical species within intact mammalian cells using secondary ion mass spectrometry, with simultaneous mapping of subcellular elemental and molecular species along with intrinsic membrane-specific cellular markers. Results from imaging of both the cell surface and cell interior exposed by site-specific focused ion beam milling demonstrate that in-plane resolutions of approximately 400 nm can be achieved. The results from mapping cell surface phosphatidylcholine and several other molecular ions present in the cells establish that spatially-resolved chemical signatures of individual cells can be derived from novel multivariate analysis and classification of the molecular images obtained at different m/z ratios. The methods we have established for specimen preparation and chemical imaging of cell interiors provide the foundation for obtaining 3D molecular maps of unstained mammalian cells, with particular relevance for probing the subcellular distribution of small molecules, such as drugs and metabolites.