Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection

Abstract

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP 2 -H and AnP 2 -OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,Ndialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP 2 -H and AnP 2 -OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP 2 -OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules

“… [70] Although not covered here in detail, other rotor dyes have also been used to investigate the microviscosity of lipid membranes and intracellular compartments; through intensity, ratiometric, or lifetime‐based measurements. These include merocyanine, [112] anthracene, [113] triphenylamine, [114] juliodine,[ 32 , 33 ] porphyrin,[ 45a , 46b ] pyrrolic compounds, [115] GFP, [87a] or indocarbocyanine nanoparticle [116] —based molecular rotors.…”