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

• Type of Material: Article
• Authors: Aaron T Blanchard, Emory University; Joshua M Brockman, Emory University; Khalid Salaita, Emory University; Alexa L Mattheyses, University of Alabama at Birmingham
• Language: English
• Date: 2020-03-30
• Publisher: Optica Publishing Group
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1364/OE.381676
• Title of Journal or Parent Work: Optics Express
• Volume: 28
• Issue: 7
• Start Page: 10039
• End Page: 10061
• Grant/Funding Information: National Institutes of Health10.13039/100000002 (R01 AR072697;, RO1 GM124472;, R01 GM131099); National Science Foundation10.13039/100000001 (1350829;, CAREER 1553344;, CAREER 1832100;, DGE-1444932;, IDBR 1353939).
• Keywords: Fluorescence; Algorithms; Spectrum Analysis; Monte Carlo Method; Fluorescent Dyes; Imaging, Three-Dimensional; Photons
• Research Categories: Chemistry, General; Biology, Cell; Engineering, Biomedical

Abstract

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.