Fluorescence ratiometry and fluorescence lifetime (FLIM) imaging: Dual mode imaging cellular viscosity by a single molecular rotor

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells such as ROS and RNS. Intracellular viscosity changes relate to a number of diseases and pathologies. So it is meaningful to investigate the microviscosity at cellular level. Fluorescent molecular rotors are recently developed sensors used to determine the environmental viscosity. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. Cyanine dyes are widely used in bio-imaging and bio-detection. RY with an aldehyde group (CHO) to generate large pseudo Stokes Shift, in nonviscous media, free rotation of the CHO gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous media results in dramatic fluorescence increase (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The molecular sensor is sensitive to viscosity performing dual mode response to the variation of viscosity. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and FLIM. Although living cells are complex, the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.