Single Molecule Wide-Field Microscope with Light Sheet Illumination

亚细胞结构的动态可视化提供了一个重要的工具来解释基本的生物过程，如细胞周期、蛋白质相互作用、区隔化或运输。最近的方法进步使得光学分辨率低于光衍射的极限，在20 - 120纳米的范围内。虽然点点法和STED可以分辨20nm的结构，但这些方法只允许在特定情况下进行活细胞成像，即使用原核细胞，使用特定的染料和定制的设置。然而，SIM是一种宽视场方法，通过结构化的照明模式实现超分辨率（120纳米），具有基于相机的快速采集，非常适合活细胞应用。因此，该方法与传统的荧光团兼容。通过将结构照明从网格模式改变为晶格模式，原始图像的数量和光毒性的数量减少，这代表了对更多光敏哺乳动物细胞的活细胞成像的关键要求。此外，与其他方法相比，Lattice SIM提供了更高的穿透深度，可以对更大的标本（如类器官）进行体积活细胞成像。该装置是唯一一款商用显微镜，实现了专有的Lattice SIM与SMLM（单分子定位显微镜），为不同的实验条件提供多功能，直接和灵活的超分辨率显微镜。这种成像平台将能够对基础生物过程进行高分辨率的时空表征，例如核肌动蛋白或单分子动力学，这些过程以前很难通过传统显微镜处理。

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.