Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ExSTED to be resolved. Our work provides a robust template for super-resolution microscopy of entire cells in the ten nanometer range.

中文翻译：

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膨胀刺激发射耗尽显微镜（ExSTED）

与衍射极限成像相比，通常使用受激发射耗竭（STED）显微镜来分辨细胞的超微结构，其分辨率高约10倍。尽管STED显微镜基于用光准备荧光探针的激发态，但最近开发的扩展显微镜（ExM）通过在显微镜检查前物理放大样品来提供亚衍射分辨率。通过水凝胶的交联和溶胀，固定细胞的膨胀很容易使样品增加约4倍，因此有效光学分辨率提高了这个倍数。为了克服这些互补方法的当前局限性，我们将ExM与STED（ExSTED）结合使用，并证明了与传统显微镜（横向<10 nm，各向同性〜50 nm）相比，分辨率提高了30倍。尽管分辨率的提高很简单，但我们发现需要通过多表位进行高保真标记才能获得能够解决ExSTED超结构细节的发射体密度。我们的工作为十纳米范围内的整个细胞的超分辨率显微镜提供了一个强大的模板。