A fundamental challenge with fluorophore orientation measurement is degeneracy, which is the inability to distinguish between multiple unique fluorophore orientations. Techniques exist for the non-degenerate measurement of the orientations of single, static fluorophores. However, such techniques are unsuitable for densely labeled and/or dynamic samples common to biological research. Accordingly, a rapid, widefield microscopy technique that can measure orientation parameters for ensembles of fluorophores in a non-degenerate manner is desirable. We propose that exciting samples with polarized light and multiple incidence angles could enable such a technique. We use Monte Carlo simulations to validate this approach for specific axially symmetric ensembles of fluorophores and obtain optimal experimental parameters for its future implementation.

中文翻译：

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可变入射角线性二向色性（VALiD）：一种用于荧光体的独特3D方向测量的技术。

荧光团取向测量的一个基本挑战是简并性，即无法区分多个独特的荧光团取向。存在用于单个静态荧光团的方向的非简并测量的技术。但是，这种技术不适用于生物学研究中常见的密集标记和/或动态样品。因此，需要一种能够以非简并方式测量荧光团的取向参数的快速，宽视野显微镜技术。我们建议用偏振光和多个入射角激发样品可以实现这种技术。我们使用蒙特卡洛模拟来验证这种方法对特定的荧光团的轴向对称集合，并获得最佳的实验参数以供将来实施。