Photoelectron microscopy and photoelectron quantum yields of the fluorescent dyes flourescein and rhodamine

Abstract

Photoelectric properties of the dyes fluorescein and rhodamine were determined with the aim of assessing the usefulness of these compounds as labels in photoelectron microscopy. The photoelectron quantum yields were measured over the wavelength range 180–230 nm. At 230 nm the quantum yields for fluorescein disodium salt, rhodamine B free base and rhodamine B HCl salt are approximately 10 -5 electrons per incident photon. At 180nm these values rise to approximately 10 -3 electrons per incident photon. All forms of fluorescein do not have the same quantum yield. The neutral form of fluorescein has a quantum yield an order of magnitude lower than the disodium salt. Beam current measurements were performed on labeled and unlabeled proteins to determine the effect of the high light intensity employed in the photoelectron microscope. The initial beam current measurements and the quantum yield curves are consistent and demonstrate that there is significant contrast between labeled and unlabeled proteins. However, after several minutes in the photoelectron microscope, the proteins become more photoemissive and the contrast diminishes. This change in contrast explains several puzzling observations in the literature.