Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2022;2440:253-270. doi: 10.1007/978-1-0716-2051-9_15.
Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.
受激发射耗竭（STED）显微镜是一种光学超分辨率显微镜（SRM）技术，最近也被称为纳米显微镜，在过去十年中在生物学家中变得流行。这些技术不断将光学分辨率的物理极限推向分子尺度。从而，它们使生物学家能够以以前仅使用电子显微镜才能达到的几乎分子细节水平对细胞和组织结构进行成像。同时，它们保留了光学显微镜的优势，特别是在样品制备和成像灵活性方面。商业上可用的 SRM 设备变得越来越可用，并且在光学性能方面变得越来越复杂，重要的是，使用起来也越来越简单。机构显微镜核心设施现在广泛提供了对这种类型系统的访问。然而，该领域发展如此之快，以至于生物学家很容易被众多可用技术和方法所淹没。在这种广泛的 SRM 模式中，STED 在一个方面脱颖而出：它本质上是高级共聚焦显微镜的扩展。与其他一些 SRM 技术相比，大多数经验丰富的共聚焦显微镜用户会发现过渡到 STED 显微镜相对容易。这也适用于 STED 样品制备。尽管如此，由于 STED 显微镜中的分辨率不仅取决于入射光的波长和物镜的数值孔径，而且还取决于耗竭激光的强度的平方根，并且通常取决于荧光团与耗竭激光的光化学反应，因此在 STED 样品制备中需要一些额外的考虑因素。在这里，我们描述了 somatostatin receptor subtype 2A (SSTR2A) 的单染和 trans-Golgi-network protein TGN 38 和 t-SNARE syntaxin-6 的双色染色用于内分泌细胞系 AtT20 的 STED 和样品的 STED 成像，提供尽可能通用的协议。在这个章节中，这些协议以这种方式在机构显微镜核心设施中使用。
