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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

基本信息

批准号：
10707059
负责人：
JOHN A MAROHN
金额：
$ 30.41万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
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/10707059
关键词：
3-Dimensional Acceleration Antibodies Architecture Biological Biological Process Biology Cells Collection Complex Data Detection Development Plans Diameter Disease Electrons Explosion Freezing Frequencies Frustration 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 ultra high resolution

项目摘要

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.

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。