Fluorescence lifetime microscopy reveals the biologically-related photophysical heterogeneity of oxyblepharismin in light-adapted (blue) Blepharisma japonicum cells.

The step-up photophobic response of the heterotrich ciliate Blepharisma japonicum is mediated by a hypericinic pigment, blepharismin, which is not present in any of the known six families of photoreceptors, namely rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. Upon irradiation, native cells become light-adapted (blue) by converting blepharismin into the photochemically stable oxyblepharismin (OxyBP). So far, OxyBP has been investigated mainly from a photophysical point of view in vitro, either alone or complexed with proteins. In this work, we exploit the vivid fluorescence of OxyBP to characterize its lifetime emission in blue B. Japonicum cells, on account of the recognized role of the fluorescence lifetime to provide physicochemical insights into the fluorophore environment at the nanoscale. In a biological context, OxyBP modifies its emission lifetime as compared to isotropic media. The phasor approach to fluorescence lifetime microscopy in confocal mode highlights that fluorescence originates from two excited states, whose relative balance changes throughout the cell body. Additionally, Cilia and kinetids, i.e., the organelles involved in photomovement, display lifetime asymmetry between the anterior and posterior part of the cell. From these data, some hypotheses on the phototransduction mechanism are proposed.