Cell Biology and Biophysics, European Molecular Biology Laboratory, Heidelberg, Germany.
Institute of Applied Optics and Biophysics, Friedrich-Schiller-University, Jena, Germany.
Nat Commun. 2021 Feb 19;12(1):1180. doi: 10.1038/s41467-021-21333-x.
3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.
3D 单分子定位显微镜（SMLM）是一种新兴的结构细胞生物学超分辨率方法，因为它可以探测细胞结构中蛋白质的精确位置。在超临界角定位显微镜（SALM）中，单荧光团的 z 位置是从超临界荧光强度中提取出来的，而超临界荧光强度强烈依赖于它们与盖玻片的距离。在这里，我们通过直接分离超临界和亚临界发射，使用超高数值孔径物镜，并应用拟合程序提取单发射器的精确强度，充分发挥了 SALM 的潜力，并将其 z 分辨率提高了四倍以上。我们在 DNA 折纸结构以及细胞中的网格蛋白包被小泡和微管上证明了纳米各向同性定位精度，说明了 SALM 在细胞生物学中的潜力。
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