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.

概括确定在10至70的细胞中荧光标记的生物分子的三维位置的能力 NM分辨率导致了生物学发现的爆炸。超分辨率光学显微镜已导致最近在我们对各种蛋白质组件中分子组织的理解中的戏剧性突破并导致了细胞器中存在的新超分子体系结构的发现。空间分辨率通常令人沮丧的是，通过超分辨率的光学显微镜保留，认为比大多数生物分子更大。该技术开发建议的目标是创建一种用于本地化单个生物分子的技术具有Angstrom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acton敏感性悬臂为100 在高温温度下，在高真空中，纳米直径磁尖在样品表面附近操作。磁铁尖式悬臂扮演两个角色。它充当力梯度探测器，使得能够观察从单个电子旋转的磁共振作为悬臂机械共振频率的转移。它此外，还提供了磁场梯度，5高斯/埃斯特罗姆或更大的磁场梯度，这使得三个具有Angstrom空间分辨率的单个电子自旋标记的尺寸磁共振成像。证明- 概念数据的数据已获得，证明了从100的硝基氧化物中检测磁共振的能力自旋标签并以比直径小100倍的分辨率以空间解析电子自旋密度磁性尖端。我们提出了一项逐步的技术开发计划 - 以理论，模拟和初步数据为支持 - 用于实现单个氮氧化物自旋标签的检测并在三个维度上成像其位置具有Angstrom精度。拟议的创新包括通过在高处运行来实现近乎统一的自旋极化使用新型的低温芯片尺度微波源，磁场和低温，采用更好的间悬臂位置探测器和自旋调制方案，以逃避样品相关的噪声，并利用同步的悬臂和自旋兴奋脉冲序列以实现高纤维旋转调制，发展强大的贝叶斯图像收集和重建协议，并制造改进的悬臂和磁性提高人旋转灵敏度的提示。该技术将使用良好的核酸规则来验证，生物分子，蛋白质复合物和抗体。概念证明实验将进行证明该技术对烟灰冷冻的生物样品的适用性以及执行相关性易变的能力恢复定位实验。一起进行拟议的工作代表了一种用于本地化的新技术在具有Angstrom精确度的粉状细胞中，个体（自旋标记和富含标记的）生物分子。