Single-molecule localization microscopy is a powerful tool for visualizing subcellular structures, interactions and protein functions in biological research. However, inhomogeneous refractive indices inside cells and tissues distort the fluorescent signal emitted from single-molecule probes, which rapidly degrades resolution with increasing depth. We propose a method that enables the construction of an in situ 3D response of single emitters directly from single-molecule blinking datasets, and therefore allows their locations to be pinpointed with precision that achieves the Cramér-Rao lower bound and uncompromised fidelity. We demonstrate this method, named in situ PSF retrieval (INSPR), across a range of cellular and tissue architectures, from mitochondrial networks and nuclear pores in mammalian cells to amyloid-β plaques and dendrites in brain tissues and elastic fibers in developing cartilage of mice. This advancement expands the routine applicability of super-resolution microscopy from selected cellular targets near coverslips to intra- and extracellular targets deep inside tissues.

中文翻译：

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全细胞和组织的三维纳米显微镜，具有原位点扩展功能检索。

单分子定位显微镜是在生物学研究中可视化亚细胞结构，相互作用和蛋白质功能的强大工具。但是，细胞和组织内部的折射率不均匀会扭曲从单分子探针发出的荧光信号，这会随着深度的增加而迅速降低分辨率。我们提出了一种方法，该方法能够直接从单分子闪烁数据集中构建单个发射器的原位3D响应，因此可以精确定位其位置，从而实现Cramér-Rao下界和不折不扣的逼真度。我们演示了这种方法，该方法在一系列细胞和组织结构中都被称为原位PSF检索（INSPR），从哺乳动物细胞的线粒体网络和核孔到大脑组织中的淀粉样蛋白-β斑块和树突以及正在发育的小鼠软骨中的弹性纤维。这一进步将超分辨率显微镜的常规应用范围从盖玻片附近的选定细胞靶扩展到了组织内部深处的细胞内和细胞外靶。