Here we apply the SUPPOSe algorithm on images acquired using Stimulated Emission Depletion (STED) microscopy with the aim of improving the resolution limit achieved. We processed images of the nuclear pore complex (NPC) from cell lines in which the Nup96 nucleoporin was endogenously labeled. This reference protein forms a ring whose diameter is ∼107 nm with 8 corners ∼42 nm apart from each other. The stereotypic arrangement of proteins in the NPC has been used as reference structures to characterize the performance of a variety of microscopy techniques. STED microscopy images resolve the ring arrangement but not the eightfold symmetry of the NPC. After applying the SUPPOSe algorithm to the STED images, we were able to solve the octagonal structure of the NPC. After processing 562 single NPC, the average radius of the NPC was found to be R = 54.2 ± 2.9 nm, being consistent with the theoretical distances of this structure. To verify that the solutions obtained are compatible with a NPC-type geometry, we rotate the solutions to optimally fit an eightfold-symmetric pattern and we count the number of corners that contain at least one localization. Fitting a probabilistic model to the histogram of the number of bright corners gives an effective labeling efficiency of 31%, which is in agreement with the values reported in for other cell lines and ligands used in Single Molecule Localization microscopy, showing that SUPPOSe can reliably retrieve sub-resolution, nanoscale objects from single acquisitions even in noisy conditions.

中文翻译：

---

使用 SUPPOSe 改进 STED 显微镜：增强单图像的分辨率

在这里，我们将 SUPPOSe 算法应用于使用受激发射损耗 (STED) 显微镜采集的图像，目的是提高所达到的分辨率极限。我们处理了来自 Nup96 核孔蛋白内源标记的细胞系的核孔复合体 (NPC) 图像。该参考蛋白形成一个直径约为 107 nm 的环，有 8 个角，彼此相距 42 nm。 NPC 中蛋白质的定型排列已被用作参考结构来表征各种显微镜技术的性能。 STED 显微镜图像解析了 NPC 的环排列，但无法解析八重对称性。将 SUPPOSe 算法应用于 STED 图像后，我们能够求解 NPC 的八边形结构。处理562个单个NPC后，发现NPC的平均半径为R = 54.2±2.9 nm，与该结构的理论距离一致。为了验证所获得的解是否与 NPC 类型的几何体兼容，我们旋转解以最佳地拟合八重对称图案，并计算包含至少一个定位的角的数量。将概率模型拟合到亮角数量直方图可得到 31% 的有效标记效率，这与单分子定位显微镜中使用的其他细胞系和配体报告的值一致，表明 SUPPOSe 可以可靠地检索即使在嘈杂的条件下，也可以通过单次采集获得亚分辨率、纳米级物体。