Abstract：
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.
摘要：
受激发射损耗（STED）显微镜是光学超分辨率显微镜（SRM）技术之一，最近也被称为纳米显微镜，在过去的十年里，它在生物学家中越来越受欢迎。这些技术不断将光学分辨率的物理边界推向分子尺度。因此，它们使生物学家能够在几乎分子细节的水平上对细胞和组织结构进行成像，这在以前只能使用电子显微镜来实现。一直以来,它们保留了光学显微镜的优点，特别是关于样品制备和成像的灵活性。商业上可用的SRM设置变得越来越可用，也越来越复杂，在光学性能和，重要的是,易用性。机构显微镜核心设施现在提供了对这种类型系统的广泛访问。然而,这个领域发展如此迅速，并不断增长，生物学家很容易被众多可用的技术和方法所淹没。从大量的SRM模式中，STED在一个方面脱颖而出：它本质上是对先进的共聚焦显微镜的扩展。与其他一些SRM技术相比，大多数有经验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，因为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，但至关重要的是，损耗激光强度的平方根，总的来说,关于荧光团与耗尽激光的光化学相互作用，在STED样品制备中需要一些额外的考虑。在这里，我们描述了生长抑素受体亚型2A（SSTR2A）的单色染色和跨高尔基网络蛋白TGN38和t-SNAREsyntaxin-6在内分泌细胞系AtT20和STED成像样品中的STED的双色染色，以尽可能一般的形式提供协议。本章中的协议以这种方式在机构显微镜核心设施中使用。
