Single molecular localization microscopy (SMLM) is a useful tool in biological observation with sub-10-nm resolution. However, SMLM is incapable of discerning two molecules within the diffraction-limited region unless with the help of a stochastic on–off switching scheme which yet entails time-consuming processes. Here, we produce a novel kind of focal spot pattern, called sub-diffraction dark spot (SDS), to localize molecules within the sub-diffraction region of interest. In our proposed technique nominated as sub-diffracted dark spot localization microscopy (SDLM), multiple molecules within the diffraction-limited region could be distinguished without the requirement of stochastic fluorescent switches. We have numerically investigated some related impacts of SDLM, such as detection circle diameter, collected photon number, background noise, and SDS size. Simulative localization framework has been implemented on randomly distributed and specifically structured samples. In either two- or three-dimensional case, SDLM is evidenced to have $\sim 2\mathrm{nm}$ localization accuracy.

中文翻译：

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亚衍射暗点定位显微镜

单分子定位显微镜 (SMLM) 是一种具有亚 10 纳米分辨率的生物观察工具。然而，SMLM 无法识别衍射极限区域内的两个分子，除非借助随机开关方案，但需要耗时的过程。在这里，我们产生了一种新型的焦点图案，称为亚衍射暗点 (SDS)，以将分子定位在感兴趣的亚衍射区域内。在我们提出的称为亚衍射暗点定位显微镜 (SDLM) 的技术中，可以区分衍射限制区域内的多个分子，而无需随机荧光开关。我们已经数值研究了 SDLM 的一些相关影响，例如探测圆直径、收集的光子数、背景噪声、和 SDS 大小。模拟定位框架已在随机分布和特定结构的样本上实施。在二维或三维情况下，SDLM 被证明具有$～2\mathrm{纳米}$ 定位精度。