Styryl dyes for viscosity measurement and detection of pathological processes in mitochondria of living cells using fluorescence lifetime imaging microscopy, a critical study

In the present work, two molecular rotors based on styryl dyes were synthesized and investigated. Both dyes showed significant sensitivity to the viscosity of the medium. Due to their significant positive charge, they efficiently accumulated in mitochondria of living cells. Binding to mitochondria resulted in increased fluorescence intensity and fluorescence lifetime of the dyes, allowing them to be visualized without washing off the unbound dye. We have also shown that the fluorescence lifetime of the studied molecular rotors is determined not only by the viscosity of the medium, but also by interactions with cell components such as proteins and nucleic acids. The present work clearly shows that these interactions do not allow a reliable estimation of viscosity in cell organelles using the synthesized dyes. This compromises the results of previous viscosity studies using molecular rotors at least of the styryl type. Nevertheless, induction of cell apoptosis by benzylviologen led to an increase in brightness and fluorescence lifetime of the dyes, which can be caused by both changes in viscosity and changes in the expression profile of proteins localized in mitochondria and interacting with the dyes. Thus, the obtained styryl dyes, like previously published homologous compounds, cannot be used for viscosity measurements, but can be used for identification of pathological states of the cell.