Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation of the imaging results. STED microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Because these factors are related to both the sample and the system, a reliable estimation of the resolution is not straightforward. Here we show a method based on the Fourier ring correlation (FRC), which estimates an absolute resolution value directly from any STED and, more in general, point-scanning microscopy image. The FRC-based resolution metric shows terrific sensitivity to the image signal-to-noise ratio, as well as to all sample and system dependent factors. We validated the method both on commercial and on custom-made microscopes. Since the FRC-based metric can be computed in real time, without any prior information of the system/sample, it can become a fundamental tool for (i) microscopy users to optimize the experimental conditions and (ii) microscopy specialists to optimize the system conditions.

中文翻译：

---

在受激发射耗尽显微镜中评估图像分辨率

准确了解激发发射耗尽（STED）显微镜实验中的有效空间分辨率对于可靠解释成像结果至关重要。STED显微镜理论上提供了分子分辨率，但实际上不同的因素限制了它的分辨率。由于这些因素与样品和系统都有关，因此对分辨率进行可靠的估算并不是一件容易的事。在这里，我们展示了一种基于傅立叶环相关（FRC）的方法，该方法可以直接从任何STED以及更通常的点扫描显微镜图像中估算绝对分辨率值。基于FRC的分辨率指标显示出对图像信噪比以及所有样本和系统相关因素的极高灵敏度。我们在商业显微镜和定制显微镜上均对该方法进行了验证。