Highly multiplexed ion-beam tissue molecular imaging with sub-micron resolution

描述(由申请人提供)：能够在保存形态的细胞和组织中同时可视化许多蛋白质的存在、丰度、位置和功能状态一直是一个目标。这项建议旨在优化最近展示的基于质谱仪的成像方法，当完全开发时，可以使用同时应用的质量标签标记抗体来检测细胞和组织中多达40到100+个分子目标，并具有出色的形态。这项技术类似于激光扫描共聚焦显微镜，不同的是扫描是使用紧密聚焦的离子束，而不是类似大小的激光光点。检测到的标记不是荧光团，而是附着在任何给定探针上的金属原子团。使用高聚焦扫描氧离子束和10种抗体同时应用于临床乳腺癌组织的初步数据显示，组织学相似的图像揭示了结合抗体的亚细胞分辨率定位。展望未来，我们希望优化灵敏度和可靠性，目标是使其成为基础和翻译科学发现、药物开发和可能的临床部署的标准、高性能、成像方法。本项目将涉及：用于蛋白质分析物的可靠的分析前样品制备方法和标记技术；基于当前和预测仪器的离子束成像方法改进；用于设备控制、显示和分析的功能强大、易于使用的软件；以及在几个重要的生物测试案例中的验证。

DESCRIPTION (provided by applicant): The ability to visualize the presence, abundance, location and functional state of many proteins at once in cells and tissues with preserved morphology has long been a goal. This proposal seeks to optimize recently demonstrated mass-spec-based imaging methods that can, when fully developed, detect as many as 40 to 100+ molecular targets in cells and tissues using simultaneously applied mass-tag-labeled antibodies, with outstanding morphology. The technique is analogous to laser-scanning confocal microscopy, except that the scanning is performed using a tightly focused ion beam, rather than a similarly sized point of laser light. And instead of fluorophores, the labels detected are clustes of metal atoms attached to any given probe. Preliminary data using a highly focused, scanning oxygen ion beam and 10 antibodies applied simultaneously to clinical breast cancer tissues demonstrate histology-like images that reveal bound antibody location with subcellular resolution. Going forward, we would like to optimize sensitivity and reliability, with the goal of making this a standard, high-performance, imaging approach for basic and translational science discovery, drug development, and possible clinical deployment. The present project will address: reliable pre-analytical sample preparation methods and labeling techniques for protein analytes; ion-beam imaging method improvements based on current and projected instrumentation; powerful, easy-to-use software for equipment control, display and analysis; and validation in several important biological test cases.