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.

总结 用10到70个细胞的荧光标记的生物分子的三维位置的能力， 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导致了最近的 我们对各种蛋白质组装体中分子组织的理解取得了重大突破 并导致发现了细胞器中存在的新的超分子结构。空间分辨率通常 令人沮丧的是，通过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。 这项技术开发提案的目标是创造一种定位单个生物分子的技术 精确到埃。我们提出了一种技术，使用自旋标签定位分子。拟议工作 将采用磁共振力显微镜，其中具有100 纳米直径的磁性尖端在低温下的高真空中在样品表面附近操作。 磁铁悬臂有两个作用。它作为一个力梯度探测器，能够观察到 磁共振从个别电子自旋作为悬臂的机械共振频率的偏移。它 此外，还提供了5高斯/埃或更大的磁场梯度源，这使得三个 具有埃空间分辨率的单个电子自旋标记的三维磁共振成像。证据- 已经获得了概念数据，证明了从100个氮氧化物中检测磁共振的能力 自旋标记，并在空间上分辨电子自旋密度的分辨率小于100倍的直径， 磁性尖端。 我们提出了一个逐步的技术开发计划-由理论，模拟和初步数据支持 - 为了实现单个氮氧自旋标记的检测并在三维中成像它们的位置 精确到埃。所提出的创新包括通过在高的温度下操作来实现接近统一的自旋极化。 磁场和低温使用新的低温芯片级微波源，采用更好的内部， 用于避免样品相关噪声的悬臂梁位置检测器和自旋调制方案， 同步的悬臂梁和自旋激发脉冲序列，以实现高保真度的自旋调制， 强大的贝叶斯图像收集和重建协议，并制造改进的杠杆和磁 提高每次旋转灵敏度的提示。该技术将使用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， 该技术对冷冻生物样品的适用性和进行相关检测的能力， 定位实验。总之，拟议的工作代表了一种新的技术， 单个（自旋标记的和荧光标记的）生物分子以埃的精度在螺旋桨冷冻的细胞中。