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

Abstract

5Xanthene 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 10 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.

Linear dichroism of visible-region chromophores using M13 bacteriophage as an alignment scaffold

“…When Cy3 was deposited onto PE ox after it had been stretched, the LD spectrum varied with stock solution concentration and contained bands that did not always correlate with the absorbance spectra: there were two negative bands at 505 nm and 547 nm and two positive bands at 445 nm and 585 nm in the LD spectra and just two bands at 527 nm and 570 nm in the absorbance spectra (data not shown) with different shaped spectra being observed for different amounts of sample deposited. The inconsistency of the wavelength and number of bands in the absorbance and LD spectra is consistent with the formation of higher order (at least dimeric) dye oligomers, albeit of unknown packing mode 36. When 30 μL of 0.025 mg mL –1 Cy3 was deposited onto un-stretched PE ox and then stretched after the dye had dried (Fig.…”

Chiroptical detection of self‐aggregating fluorescent rhodamine conjugates: Mistakes and prospects

“…The transition moment of the main visible absorption band of RhB is polarized along the long axis of the xanthene nucleus. 23 Applying this rule, the longer-wavelength positive and the shorter-wavelength negative CE obtained for T 3 -RhB (Figure 2) predict that the long axes of proximal xanthene units form a positive (right handed) twist angle inside the assembly.…”