Targetable and fixable rotor for quantifying mitochondrial viscosity of living cells by fluorescence lifetime imaging

It is meaningful to accurately quantify the changes in local viscosity within the mitochondria of living cells, because viscosity influences mitochondrial network organization and metabolite diffusion. Although many molecular probes targeting mitochondria have been reported, almost all of them are not fixed to the mitochondria. Thus, they may not be suitable for sensing in abnormal mitochondria with lowered potential. In order to monitor viscosity in all mitochondria, no matter their working or health status, we develop the first fixable BODIPY (boron-dipyrromethene) rotor, named Vis-A. Vis-A contains an aldehyde group as an anchor to react with amino groups of proteins, which make it immobilizable in mitochondria. Vis-B, the reference compound without such anchor unit, is also synthesized. Both Vis-A and Vis-Bshow excellent mitochondrial targetability, as good as the commercially available mitochondrial dye (Mito Tracker Deep Red). However, when cells are chemically treated to decrease the mitochondrial potential, only Vis-A continues emitting strong fluorescence in mitochondria, but the signals of Vis-B and Mito Tracker Deep Red completely disappear. This comparison confirms that Vis-A not only specifically localizes in mitochondria, but also can be stably retained there. Then, Vis-A is applied to detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A, the changes in viscosity under typical pathological conditions (i.e., treatment with rotenone and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) for mitochondria are monitored by FLIM.