Biology Department, Syracuse University, Syracuse, NY, USA.
Methods Mol Biol. 2022;2364:3-24. doi: 10.1007/978-1-0716-1661-1_1.
Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.
近年来，超分辨率（SR）成像技术发展迅速，但只有其中的一部分技术足够温和，可以被细胞生物学家用来研究活细胞，同时将光损伤降到最低。我们的研究集中在研究胰岛素分泌过程中活胰腺β细胞中肌动蛋白细胞骨架的动态重塑。这些研究需要超分辨率的光显微镜技术，这些技术足够温和，可以实时记录肌动蛋白细胞骨架的快速变化。在这一章中，我们描述了一种突破传统光显微镜衍射极限的 SR 技术，称为 TIRF-SIM。使用这种 SR 技术，我们已经能够证明（1）胰腺β细胞上的微绒毛在质膜平面上迁移，以及（2）当细胞被刺激分泌胰岛素时，皮质肌动蛋白网络重新组织。我们描述了用于处理和分析 TIRF-SIM 图像以获得定量数据的 FIJI 插件。
