Variable incidence angle linear dichroism (VALiD): a technique for unique 3D orientation measurement of fluorescent ensembles

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. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement