Single Molecule Wide-Field Microscope with Light Sheet Illumination

带光片照明的单分子广视野显微镜

• 批准号: 437835311
• 金额: --
• 依托单位: Albert-Ludwigs-Universität Freiburg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/437835311?language=en
• 关键词: Single; Molecule; Wide; Field; Microscope

The dynamic visualization of subcellular structures provides an essential tool to explain fundamental, biological processes, such as the cell cycle, protein interactions, compartmentalization or transport. Recent methodological advances enabled optical resolutions below the limit of light diffraction, in a range of 20 – 120 nm. While pointillism and STED resolve structures down to 20 nm, these methods only allow live cell imaging in specialized contexts, i.e. with prokaryotic cells and using specific dyes and custom made setups. SIM however is a wide-field method achieving super-resolution (120 nm) by structured illumination patterns with fast camera-based acquisition ideal for live cell applications. As such, this method is compatible with conventional fluorophores. By changing the structured illumination from a grid to a lattice pattern, the number of raw images and thereby the amount of phototoxicity is reduced, which represents a critical requirement for live cell imaging of more photosensitive mammalian cells. Further, Lattice SIM offers a higher penetration depth than other approaches, enabling volumetric live cell imaging of larger specimen, such as organoids. The setup is the only commercially available microscope implementing the proprietary Lattice SIM with SMLM (single molecule localization microscopy) offering versatile, straight forward and flexible super-resolution microscopy for different experimental conditions. Such imaging platform shall enable the high resolution spatiotemporal characterization of fundamental biological processes such as for example nuclear actin or single molecule dynamics, which were previously difficult to tackle by conventional microscopy.

亚细胞结构的动态可视化提供了解释基本生物过程（例如细胞周期、蛋白质相互作用、区室化或运输）的重要工具。最近的方法论进展使光学分辨率低于光衍射极限，范围为 20 – 120 nm。虽然点画法和 STED 可以解析低至 20 nm 的结构，但这些方法仅允许在特殊环境下进行活细胞成像，即使用原核细胞并使用特定染料和定制设置。然而，SIM 是一种宽视场方法，通过结构化照明模式和基于相机的快速采集实现超分辨率 (120 nm)，非常适合活细胞应用。因此，该方法与常规荧光团兼容。通过将结构化照明从网格改为点阵图案，原始图像的数量减少，从而减少光毒性，这代表了对光敏性更强的哺乳动物细胞的活细胞成像的关键要求。此外，Lattice SIM 提供比其他方法更高的穿透深度，能够对较大样本（例如类器官）进行体积活细胞成像。该装置是唯一一款采用带有 SMLM（单分子定位显微镜）的专有 Lattice SIM 的商用显微镜，为不同的实验条件提供多功能、直接且灵活的超分辨率显微镜。这种成像平台将能够对基本生物过程（例如核肌动蛋白或单分子动力学）进行高分辨率时空表征，这在以前很难通过传统显微镜来解决。