Optical properties of xanthene based fluorescent dyes studied by stretched-film linear dichroism

Abstract

Xanthene dyes are commonly used to label proteins in order to probe their location and activity using fluorescence spectroscopy and microscopy. However, fundamental properties such as the polarizations of transitions for many of the dyes have not been available. In this paper we report the use of recently developed oxidised polyethylene (PE^OX) stretched film linear dichroism to determine the transition polarizations of xanthene, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyronin Y, pyronin B, fluorescein, and rhodamine 6G. The effect of the formation of higher order structures is also discussed when they occur. The dyes (except xanthene) all have an intense long-axis polarized transition in the region of 500 nm. They also have long-axis (//) polarized transitions from about 280 nm downwards in wavelength. There are suggestions of a weak short axis (⊥) polarized transition in the region of 320–350 nm in each case.