Using rotation of linearly polarized illumination from different directions enables reconstructing the full 3D orientation of single molecules. Theoretically, already two projections suffice for a full 3D orientation determination if the intensities are properly calibrated and in the absence of noise. The question arises if a limited set of projections that cannot excite all molecules equally contains already the maximum information on the 3D orientation when also considering photon and experimental noise, or if detecting more projections or even the full angular projection space provides more information. To answer this question, we constructed a device exploring the full angular excitation space available with a distinct microscope objective and tested several different polarization modulation schemes using simulated as well as experimental single molecule data. For noisy experimental data, we found three projections ideal and no significant improvement in angular precision for larger projection numbers or when detecting the full projection space.

使用来自不同方向的线偏振照明的旋转能够重构单个分子的完整3D方向。从理论上讲，如果强度经过正确校准且没有噪声，则已经有两个投影就足以确定完整的3D方向。如果在考虑光子和实验噪声时，如果有限的一组无法激发所有分子的投影都已经包含了有关3D方向的最大信息，或者检测到更多的投影甚至整个角度的投影空间都提供了更多的信息，就会出现问题。为了回答这个问题，我们构建了一种设备，可利用独特的显微镜物镜探索可用的全角激发空间，并使用模拟和实验单分子数据测试了几种不同的偏振调制方案。对于嘈杂的实验数据，我们发现三个投影比较理想，并且对于较大的投影数量或检测到完整的投影空间时，角度精度没有明显改善。