Microspectrofluorometry, fluorescence imaging and confocal microscopy of an endogenous pigment of the marine ciliate Fabrea salina

Abstract

Fabrea salina, an apparently colorless marine ciliate in the order Heterotrichida, shows positive phototaxis and photophobic step-down reactions. Fluorescence microscopy and imaging of living cells reveal only a scarcely detectable emission, but dead cells are characterized by a strong red fluorescence, results indicating the presence of an endogenous fluorescent pigment. Single cell microspectrofluorometry, fluorescence imaging and laser confocal microscopy studies, together with time-gated fluorescence spectroscopy of cell suspensions, were performed in order to investigate the nature and localization of this pigment. The fluorescence emission spectrum at different excitation wavelenghts (from 337 to 425 nm) exhibits two peaks: the main one between 590 and about 615 nm, depending on the cell treatment, and the other one at about 655 nm. The time decay of fluorescence shows three distinct components, with lifetimes ranging from a few hundred picoseconds to some nanoseconds and relative contributions varying with the chemical treatment. Time-gated spectroscopy allows us also to observe differences in the espectral features of the distinct emitting species. Laser confocal microscopy reveals that the pigment is localized just below the cell membrane. The spectral and temporal fluorescence features of the endogenous pigment present in F. salina are very similar to the ones of blepharismin, the pigment of Blepharisma japonicum, thus suggesting that the F. salina pigment might also be a hypericin-like pigment.