Non-cleaved Electro-Mechanical Expansion (NEME) technology for super-resolution imaging of biological samples with conventional optical microscopes

非切割机电扩展 (NEME) 技术，用于使用传统光学显微镜对生物样品进行超分辨率成像

• 批准号: 10176530
• 负责人: Deblina Sarkar
• 金额: $ 24.9万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-10 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10176530
• 关键词: 3-Dimensional; Antibodies; Architecture; Benchmarking; Biological; Brain; Cells; Cellular Structures; Chemistry; Cilia; Cleaved cell; Crowding; Cultured Cells; DNA; Development; Disease; Dyes; Electron Microscopy; Electrostatics; Face; Gel; Genome; Hybrids; Image; Imaging Techniques; Imaging technology; Isotropy; Lead; Light; Mechanics; Mentors; Methodology; Methods; Microscope; Microscopy; Mus; Nucleic Acids; Nucleosomes; Optics; Outcome; Pathologic; Phase; Play; Polymer Chemistry; Polymers; Process; Proteins; Proteomics; RNA; Reagent; Research; Resolution; Role; Sampling; Signal Transduction; Specimen; Speed; Structure; Subcellular structure; Synapses; Synaptic Transmission; System; Techniques; Technology; Thick; Tissues; Transcriptional Regulation; Validation; Work; base; biological systems; brain tissue; cell type; complex biological systems; diffraction of light; experimental study; improved; insight; materials science; mechanical force; nano; nanoscale; protein complex; reconstruction; technology validation; transcriptome

Abstract Understanding the nanoscale organizations of biomolecules in complex biological systems such as the brain, can not only provide fundamental biological insights but also help in the discovery of new targets and technologies for treating diseases. Optical microscopy provides a convenient way for imaging biological samples using readily available dyes/antibodies. However, the spatial resolution of conventional optical microscopes is limited to 300 nm due to the diffraction of light waves. On the other hand, existing super-resolution optical techniques, face challenges in scalability to thick tissues and require extremely expensive hardware, which limits their application. Recently discovered expansion microscopy (ExM), which is based on physically expanding the sample (embedded in a swellable gel) by about 4.5 x and thus, achieving an effective resolution of 70 nm, is scalable and compatible with conventional optical hardware. But, its resolution of 70 nm is not sufficient for observing subcellular structures. Though the resolution can be improved through iterated ExM (iExM), it results in low biomolecular yield as it requires transfer of biomolecules from one gel to another, with the cleaving of the first gel. The proposed work aims to develop a technology for expansion, where gel cleaving or transfer of biomolecules is not required, resulting in high biomolecular yields. This technology utilizes both electrostatic and mechanical forces for expansion to achieve high expansion factors (20x to 100x), thus leading to 300 / 20 ≈ 15 nm to 300 / 100 ≈ 3 nm resolution. This technology, which I termed non-cleaved electro-mechanical expansion (NEME) is different from previous expansion technologies which utilizes only electrostatic forces for expansion. The mentored phase of the proposed work will involve the development and characterization of the NEME technology while in the independent phase, NEME will be extended for imaging of dense protein complexes as well as RNA and DNA. NEME technology can lead to super-resolution imaging without any specialized or expensive hardware and can also provide high biomolecular yields and scalability to thick tissues. Thus, it can greatly benefit simultaneous characterization of super-fine biomolecular structures and large 3D biological systems.

摘要