Sun Yonglei, Wang Quan
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892, United States.
bioRxiv. 2025 Jun 24:2025.06.17.660171. doi: 10.1101/2025.06.17.660171.
Measuring dipole orientations of fluorescent probes offers unique local structural insights of labelled biomolecules and has seen expanding applications in structural biology studies. Here we propose an alternative imaging geometry, 'dual-view' microscopy, for single-molecule dipole orientation measurements. We develop a protocol capable of measuring absorption and emission dipole orientations of single emitters. Further, through simulation, we demonstrate that absorption dipole orientation can be accurately measured with high and uniform precision in three dimensions, significantly outperforming epifluorescence microscopy. Meanwhile the emission dipole is independently narrowed down to four possible orientations and can be uniquely determined with the co-estimated absorption dipole. Dual-view microscopy represents a new paradigm in single-molecule orientation sensing and could have unique applications in imaging under cryogenic temperatures.
测量荧光探针的偶极子取向能够提供有关标记生物分子独特的局部结构信息，并且在结构生物学研究中的应用不断拓展。在此，我们提出一种用于单分子偶极子取向测量的替代成像几何结构——“双视角”显微镜。我们开发了一种能够测量单个发光体吸收和发射偶极子取向的方法。此外，通过模拟，我们证明吸收偶极子取向能够在三维空间中以高精度且均匀的精度被准确测量，显著优于落射荧光显微镜。同时，发射偶极子被独立地缩小到四个可能的取向，并且可以通过共同估计的吸收偶极子唯一确定。双视角显微镜代表了单分子取向传感的一种新范式，并且在低温成像中可能有独特的应用。
