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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：
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
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/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

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.

总结