Technology Development for 3D Electron Microscopy

我们在过去几年的工作中开发了一些新技术，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。以下概述了特别值得注意的两个领域的进展。过去一年，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尖峰和尖峰复杂结构。这是史无前例的，因为在2008年，我们通过确定前三个HIV-1尖峰复杂结构实现了突破。我们简化的实验和计算程序使我们能够全天候远程操作，数据收集的平均仪器正常运行时间为85%。在接下来的几年里，我们将继续完善这条管道，增加使用在线、无监督分类程序分离不同构象的工具。事实证明有用的一项进展是开发了数据处理策略，当三聚体Env存在于混合物中时，数据处理策略允许清楚地分离三聚体Env的闭合状态和开放状态，以及非连接和抗体连接状态。我们已经证明，识别和去除具有最低信噪比的尖峰可以提高单个尖峰之间比对的总体精度，而比对的准确性反过来决定了在评估不同种类的SIV和HIV-1混合物中Env的构象异质性时图像分类的成功。我们通过成功地分离和重建同时存在于混合物中的未连接和抗体连接的HIV-1Env复合体的不同3D结构，验证了这些计算分离程序的有效性。了解细胞内蛋白质、代谢物和元素的空间分布对于了解健康和疾病的功能具有潜在的重要意义。亚微米分辨率是定位感兴趣的亚细胞区域所必需的，而基于光学技术的技术，如荧光显微镜，已被证明对于监测细胞内和周围离散的化学变化非常有用。然而，荧光成像仅限于成像天然的荧光分子或那些特定结合在感兴趣的分析物上的分子。相比之下，基于质谱学的技术为实现同时检测多个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仪(MALDI-MS)已被广泛用于组织成像，但其典型的空间分辨率(约20-50微米)使其不适合于大多数哺乳动物细胞的亚细胞成像。基于二次离子质谱仪(SIMS)的方法，一次离子束小到50 nm到500 nm，具有更高的分辨率，因此适用于单细胞化学成像质谱学。尽管取得了这些进展，但由于与MALDI-MS相比质量范围较小(SIMS通常小于500 m/z)、较高的初级束致分子损伤水平以及目标分子的一般较低分子电离概率等几个缺点，SIMS成像仍然没有产生多少生物学发现，在亚细胞分子成像方面也只取得了一定的成功。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害(限制了生成3D数据集的能力)以及达到更好的空间分辨率的理论限制方面，也存在挑战。通过将SIMS和聚焦离子束(FIB)球磨相结合，我们在样品制备和将成像扩展到三维的策略方面都取得了进展。使用这种方法，我们使用二次离子质谱仪对完整的哺乳动物细胞内的化学物种进行了成像，同时绘制了亚细胞元素和分子物种以及固有的膜特异性细胞标记的图谱。通过定点聚焦离子束球磨曝光的细胞表面和细胞内部的成像结果表明，可以达到约400 nm的面内分辨率。绘制细胞表面磷脂酰胆碱和存在于细胞中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比下获得的分子图像进行新的多变量分析和分类来获得。我们建立的样品制备和细胞内部化学成像的方法为获得未染色的哺乳动物细胞的3D分子图提供了基础，尤其与探测小分子(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.