Single molecule localization microscopy via angstrom-scale three-dimensional imaging of electron spin labels

摘要 在10~70岁的细胞中确定fl标记生物分子的三维位置的能力 NM解决方案导致了生物学上的一大批发现。超分辨率光学显微镜导致了最近的 在我们对各种蛋白质组件中分子组织的理解上取得了重大突破 并导致细胞器中存在新的超分子结构的发现。空间分辨率通常 令人沮丧的是，由超分辨率光学显微镜实现的生物分子仍然比大多数生物分子大得多。 这项技术开发计划的目标是创造一种使单个生物分子本地化的技术 以埃级精度。我们提出了一种使用自旋标记来定位分子的技术。拟议中的工作 将使用核磁共振作用力显微镜，在显微镜中，具有100 纳米直径磁头在低温下高真空中靠近样品表面工作。 磁铁顶端的悬臂有两种作用。它起到力梯度探测器的作用，能够观察到 单个电子自转产生的磁共振是悬臂机械共振频率的变化。它 此外还提供了磁fi磁场梯度源，5高斯/埃或更大，这使得三个 具有埃空间分辨率的单个电子自旋标记物的三维磁共振成像。证明- 已经获得了概念外的数据，证明了从氮氧化物的100‘S检测磁共振的能力 自旋标记和空间解析电子自旋密度的分辨率比直径小100倍 磁性尖端。 我们提出了一个有理论、模拟和初步数据支持的逐步技术发展计划 -实现对单个氮氧化物自旋标记的检测并在三维中成像其位置 以埃级精度。提出的创新包括通过在高电压下工作来实现近乎统一的自旋极化 磁fi焊接和低温采用新型低温芯片规模的微波源，采用更好的内部 高铁测量悬臂位置检测器和自旋调制方案，以避免与样品相关的噪声，利用 同步悬臂梁和自旋激发脉冲序列实现高fi轻量级自旋调制，正在开发中 稳健的贝叶斯图像采集和重建协议，并制造改进的悬臂梁和磁力 提高每个自旋敏感度的小贴士。这项技术将使用具有良好特性的核酸尺子进行验证， 生物分子、蛋白质复合体和抗体。将进行概念验证实验，以证明 该技术对fl灰冻生物样品的适用性和进行相关fl检测的能力。 新月形本地化实验。综上所述，拟议的工作代表了一种本地化的新技术 在fl灰冻细胞中的单个(自旋标记和前述标记的)生物分子具有角精度。

Summary The ability to determine the three-dimensional location of fluorescently labeled biomolecules in cells with 10 to 70 nm resolution has led to an explosion of discoveries in biology. Super-resolution optical microscopy has led to recent dramatic breakthroughs in our understanding of the organization of molecules in a wide variety of protein assemblies and has led to discoveries of new supramolecular architectures present in organelles. The spatial resolution typically achieved by super-resolution optical microscopy remains, frustratingly, considerably larger than most biomolecules. The goal of this technology development proposal is to create a technology for localizing individual biomolecules with angstrom precision. We propose a technology for localizing molecules using spin labels. The proposed work will employ a magnetic resonance force microscope, in which an attonewton-sensitivity cantilever with a 100 nanometer diameter magnetic tip is operated near a sample surface in high vacuum at cryogenic temperatures. The magnet-tipped cantilever serves two roles. It acts as a force-gradient detector, enabling the observation of magnetic resonance from individual electron spins as a shift of the cantilever's mechanical resonance frequency. It furthermore provides a source of magnetic field gradient, 5 gauss/angstrom or larger, that makes possible the three dimensional magnetic resonance imaging of individual electron spin labels with angstrom spatial resolution. Proof- of-concept data has been acquired demonstrating the ability to detect magnetic resonance from 100's of nitroxide spin labels and to spatially resolve electron spin density at a resolution 100 times smaller than the diameter of the magnetic tip. We present a stepwise technology development plan — backed by theory, simulations, and preliminary data — for achieving the detection of individual nitroxide spin labels and imaging their locations in three dimensions with angstrom precision. Proposed innovations include achieving near-unity spin polarization by operating at high magnetic field and low temperature using novel cryogenic chip-scale microwave sources, employing better inter- ferometric cantilever position detectors and spin modulation schemes to evade sample-related noise, harnessing synchronized cantilever and spin excitation pulse sequences to achieve high fidelity spin modulation, developing robust Bayesian image collection and reconstruction protocols, and fabricating improved cantilevers and magnetic tips for increased per-spin sensitivity. The technology will be validated using well characterized nucleic-acid rulers, biomolecules, protein complexes, and antibodies. Proof-of-concept experiments will be carried out to demonstrate the applicability of the technology to flash frozen biological samples and the ability to carry out correlative fluo- rescent localization experiments. Taken together the proposed work represents a new technology for localizing an individual (spin-labeled and fluorescently labeled) biomolecule in a flash-frozen cell with angstrom precision.