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

通过电子自旋标记的埃级三维成像进行单分子定位显微镜

• 批准号: 10280393
• 负责人: JOHN A MAROHN
• 金额: $ 29.5万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10280393
• 关键词: 3-Dimensional; Antibodies; Architecture; Back; Biological; Biological Process; Biology; Caliber; Cells; Collection; Complex; Data; Detection; Development Plans; Disease; Electrons; Explosion; Freezing; Frequencies; Genetic Transcription; Goals; Image; Individual; Label; Location; Locomotion; Magnetic Resonance; Magnetic Resonance Imaging; Magnetism; Maps; Mechanics; Microscope; Microscopy; Molecular Machines; Motion; Noise; Nucleic Acids; Optics; Organelles; Physiologic pulse; Positioning Attribute; Proteins; Protocols documentation; Resolution; Role; Sampling; Scheme; Signal Transduction; Source; Spin Labels; Structure; Surface; Technology; Temperature; Three-Dimensional Imaging; Time; Translations; Vacuum; Work; cantilever; cold temperature; cryogenics; density; detector; experimental study; improved; innovation; irradiation; magnetic field; microwave electromagnetic radiation; microwave imaging; nanometer; new technology; novel; protein complex; public health relevance; reconstruction; simulation; single molecule; technology development; theories; therapy design

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.

概括 能够确定 10 至 70 个细胞中荧光标记生物分子的三维位置 纳米分辨率导致了生物学发现的爆炸式增长。超分辨率光学显微镜最近 我们对各种蛋白质组装体中分子组织的理解取得了巨大突破 并导致细胞器中新的超分子结构的发现。空间分辨率通常 令人沮丧的是，通过超分辨率光学显微镜获得的分子仍然比大多数生物分子大得多。 该技术开发提案的目标是创建一种用于定位单个生物分子的技术 以埃的精度。我们提出了一种使用自旋标签定位分子的技术。拟议的工作 将采用磁共振力显微镜，其中阿托牛顿灵敏度悬臂梁具有 100 纳米直径的磁性尖端在高真空和低温下在样品表面附近操作。 磁头悬臂有两个作用。它充当力梯度检测器，能够观察 随着悬臂机械共振频率的变化，单个电子自旋产生的磁共振。它 此外还提供了 5 高斯/埃或更大的磁场梯度源，这使得这三种方法成为可能 具有埃空间分辨率的单个电子自旋标签的三维磁共振成像。证明- 已获得概念数据，证明能够从数百个硝基氧中检测磁共振 自旋标签并以比标签直径小 100 倍的分辨率空间解析电子自旋密度 磁性尖端。 我们提出了一个逐步的技术开发计划——以理论、模拟和初步数据为支持 — 用于实现单个硝基氧自旋标签的检测并在三维空间中对它们的位置进行成像 以埃的精度。提出的创新包括通过在高电压下运行来实现近乎一致的自旋极化 磁场和低温使用新型低温芯片级微波源，采用更好的内部 铁测量悬臂梁位置探测器和自旋调制方案可避免与样品相关的噪声，利用 同步悬臂和自旋激发脉冲序列以实现高保真自旋调制，开发 强大的贝叶斯图像采集和重建协议，以及制造改进的悬臂和磁性 提高每次旋转灵敏度的技巧。该技术将使用经过充分表征的核酸标尺进行验证， 生物分子、蛋白质复合物和抗体。将进行概念验证实验来证明 该技术对闪蒸冷冻生物样品的适用性以及进行相关荧光分析的能力 最近的本地化实验。总而言之，拟议的工作代表了一种用于本地化的新技术 快速冷冻细胞中的单个（自旋标记和荧光标记）生物分子具有埃级精度。