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

摘要 了解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，但也有助于发现治疗的新靶点和技术 疾病。光学显微镜为使用现成的生物样品成像提供了一种方便的方法 染料/抗体。然而，传统光学显微镜的空间分辨率被限制在300纳米，这是由于 光波的绕射。另一方面，现有的超分辨率光学技术在可伸缩性方面面临挑战 厚厚的组织，需要极其昂贵的硬件，这限制了它们的应用。最近发现的扩展 显微镜(EXM)，这是基于将样品(嵌入在可膨胀的凝胶中)物理膨胀约4.5倍和 因此，获得70 nm的有效分辨率是可扩展的，并且与传统的光学硬件兼容。但是，它的 70纳米的分辨率不足以观察亚细胞结构。虽然分辨率可以通过以下方式提高 重复exm(IExM)，它导致低生物分子产率，因为它需要将生物分子从一种凝胶转移到另一种凝胶， 随着第一个凝胶的裂解。拟议中的工作旨在开发一种膨胀技术，在这种技术中，凝胶裂解或 不需要生物分子的转移，从而产生高生物分子产率。这项技术利用了静电和 和机械力的膨胀，以实现高膨胀系数(20x到100x)，从而导致300/20≈15 nm到 300/100≈3 nm分辨率。这项我称之为无劈裂机电膨胀(NEME)的技术 不同于以往仅利用静电力进行膨胀的膨胀技术。指导阶段 拟议工作的一部分将涉及Neme技术的开发和表征，而在 作为一种独立的相，Neme将被扩展到高密度蛋白质复合体以及RNA和DNA的成像。Neme 技术可以在不需要任何专门或昂贵的硬件的情况下实现超分辨率成像，并且还可以提供高分辨率 生物分子产量和对厚组织的可扩展性。因此，它将极大地有利于超细颗粒的同时表征 生物分子结构和大型三维生物系统。

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.