Red-Emitting Mitochondrial Probe with Ultrahigh Signal-to-Noise Ratio Enables High-Fidelity Fluorescent Images in Two-Photon Microscopy.

Herein, we reported a red-emitting probe (E)-4-(2-(8-hydroxy-julolidine-9-yl)vinyl)-1-methylpyridin-1-ium iodide (HJVPI) on a rotor mechanism with an ultrahigh signal-to-noise ratio. HJVPI could give high-fidelity fluorescent images of mitochondria in living immortalized and normal cells and be suitable for IR excitation source of two-photon microscopy and various excitation sources of confocal microscopy. As a rotor, its single/two-photon fluorescence intensities directly depended on environmental viscosity. And, as a mitochondrial probe, it displayed much larger two-photon absorption cross sections in comparison with commercial MitoTracker Green FM and MitoTracker Red FM. Moreover, the fact that living cells stained by HJVPI still possessed physiological function could also be confirmed: (1) MTT assay demonstrated that the mitochondria of cells stained retained their electron mediating ability and (2) double assay of HJVPI and SYTOX Blue nucleic acid stain (S-11348) showed that the plasma membrane of the cells stained was still intact. In addition, HJVPI possessed a number of beneficial properties in bioimaging such as good membrane permeability, high photostability, and excellent counterstain compatibility with Hoechst 33342. Related mechanism research suggested that its localization property was dependent on the mitochondrial membrane potential in living cells. All its remarkable properties can extend the investigation on mitochondria in a biological context.