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.

抽象的 了解大脑等复杂生物系统中生物分子的纳米级组织，不仅可以 提供基本的生物学见解，同时也有助于发现治疗的新靶点和技术 光学显微镜提供了一种使用现成的生物样本成像的便捷方法。 然而，由于染料/抗体的影响，传统光学显微镜的空间分辨率仅限于 300 nm。 另一方面，现有的超分辨率光学技术面临可扩展性的挑战。 厚的组织并且需要极其昂贵的硬件，这限制了它们最近发现的扩展。 显微镜 (ExM)，基于将样品（嵌入可膨胀凝胶中）物理膨胀约 4.5 倍， 因此，实现 70 nm 的有效分辨率是可扩展的并且与传统光学硬件兼容。 70 nm 的分辨率不足以观察亚细胞结构，但可以通过以下方法提高分辨率。 迭代 ExM (iExM)，它会导致生物分子产率低，因为它需要将生物分子从一种凝胶转移到另一种凝胶， 拟议的工作旨在开发一种扩展技术，其中凝胶裂解或。 该技术不需要生物分子转移，因此生物分子产率高。 和机械力进行膨胀以实现高膨胀系数（20x 至 100x），从而导致 300 / 20 ≈ 15 nm 至 300 / 100 ≈ 3 nm 分辨率，我将其称为非切割机电扩展 (NEME)。 与以前仅利用静电力进行扩展的扩展技术不同。 拟议工作的一部分将涉及 NEME 技术的开发和表征 独立阶段，NEME 将扩展到致密蛋白质复合物以及 RNA 和 DNA 的成像。 技术可以实现超分辨率成像，无需任何专门或昂贵的硬件，并且还可以提供高分辨率 因此，它可以极大地有利于超精细的同时表征。 生物分子结构和大型 3D 生物系统。