High Resolution Elemental Microscopy with the Atom Probe

许多细胞过程是由大分子组装体介导的，这些大分子组装体太大或结构变化太大而无法使用晶体学和/或NMR来解决。此外，晶体学和NMR需要将结构浓缩并从其生物环境中移除，因此阻止了在天然状态下或在其他结构的背景下进行分析。TEM具有基本的局限性，包括需要薄样品、重金属染色、图像平均化以及用于检测低原子序数元素的有限能力。我们建议开发局部电极原子探针（LEAP）来提供这些信息。使用LEAP，使用静电电离一次去除一个样品原子。通过位置敏感检测器和飞行时间质谱法确定被移除原子的位置和身份。LEAP具有<0.5 nm的位置分辨率和近量子效率，因此不需要信号平均来获得高分辨率。我们建议开发必要的样品制备方法的3-D原子级分辨率的生物大分子和大分子组装使用LEAP。最终，我们计划扩展这种能力，以提供病毒和细胞的3-D原子级结构。拟议的商业应用：LEAP在基础和临床生物学中具有商业应用，因为它能够以更高的分辨率，更完整的元素细节确定3D生物和生物分子结构，并且可能比当前的成像和分析仪器（如SEM和TEM）更快。在药物发现中，LEAP可以直接确定候选药物是否以高亲和力与靶分子结合，而不是像制药行业目前所做的那样使用分子模拟，每年花费超过2亿美元。LEAP在微电子学、材料科学和生物技术方面也有相当大的应用。

Many cellular processes are mediated by macromolecular assemblies that are too large or variable in structure to solve using crystallography and/or NMR. Moreover, crystallography and NMR require structures to be concentrated and removed from their biological milieu, therefore preventing analysis in the native state or in the context of other structures. TEM has fundamental limitations which include the need for thin samples, heavy metal stains, image averaging, and limited capabilities for detection of low atomic number elements. We propose to develop the Local Electrode Atom Probe (LEAP) to provide this information. With LEAP, sample atoms are removed one at a time using electrostatic ionization. Position and identity of removed atoms are determined by a position sensitive detector and time-of-flight mass spectrometry. LEAP has <0.5 nm positional resolution and near quantum efficiency, and therefore signal averaging is not required to obtain high resolution. We propose to develop sample preparation methods necessary for 3-D atomic-level resolution of biomacromolecules and macromolecular assemblies using the LEAP. Ultimately, we plan to extend this capability to provide 3-D atomic level structure of viruses and cells. PROPOSED COMMERCIAL APPLICATIONS: The LEAP has commercial applications in basic and clinical biology due to its ability to determine 3-D biological and biomolecular structures with higher resolution, far more complete elemental detail, and potentially more quickly than is possible with current imaging and analytical instruments such as SEMs and TEMs. In drug discovery, the LEAP can directly determine if candidate drugs bind with high avidity to target molecules, rather than to use molecular simulations as is currently done by the pharmaceutical industry, spending over $200M/yr. LEAP also has considerable applications in microelectronics, materials sciences, and biotechnology.