Construction of a ratiometric two-photon fluorescent probe to monitor the changes of mitochondrial viscosity

[Display omitted] •Response to viscosity with ratiometric fluorescence signal.•Localized in Mitochondria with a high colocalization coefficient.•Quantitative analysis of mitochondrial viscosity changes.•Ratiometric imaging viscosity in living tissues with two-photon excitation. Abnormal viscosity variations in the mitochondrial matrix may induce changes in the mitochondrial network organization and further influence metabolite diffusion, which, consequently, are related to cellular aspects of several diseases and malfunctions, such as cell malignancy, atherosclerosis, diabetes, and Alzheimer’s disease. Studies have shown that mitochondrial viscosity changes are influenced by many factors, including disruption of ionic balance by drugs, over-production of reactive oxygen species, and cell apoptosis. However, the quantization of mitochondrial viscosity changes caused by these factors is uncertain, mainly because of the lack of a suitable fluorescent viscosity probe to accurately analyze changes in mitochondrial viscosity. By combining two fluorophores that have different sensitivities to viscosity, we designed and synthesized a new mitochondrial-targeted two-photon fluorescent viscosity probe (TM-V). The probe showed a direct linear relationship between the fluorescence intensity ratio of the two fluorophores and the media viscosity, which allowed us to determine the average mitochondrial viscosity in living cells. Using the novel TM-V probe, changes in mitochondrial viscosity under various conditions were quantified. In addition, as an excellent two-proton rotor probe, TM-V was able to report on viscosity in living cells and deeper tissues by ratiometric fluorescence imaging.