Highly selective mitochondrial probes based on fluorinated pentamethinium salts: On two-photon properties and microscopic applications

Fluorescent dyes and probes have been of interest in the fields of chemistry and biology for a long time. However, with more recent applications in advanced techniques, such as two-photon and super-resolution fluorescence microscopy, higher demands are being placed on physico-chemical properties of such compounds. Another requirement is specific intracellular targeting which often remains a big challenge. Here, we present the synthesis of a series of far red-emitting fluorescent dyes based on a structure of fluorinated symmetrical ɣ-aryl substituted pentamethinium salts. We reveal the relationship between the structure and photophysical and biological properties of the compounds; a type of a side unit (indole, benzothiazole) and presence of fluorine atoms were investigated. It was found that substitution of benzothiazolium side units for indolium ones led to increased quantum yields and higher photostability of the dyes and that the presence of fluorine on the γ-aryl moiety did not exhibit significant influence on their photophysical properties. Regardless of the structural difference, all compounds localized in mitochondria of various cancerous and noncancerous cell lines and can be utilized in live-cell imaging using wide-field, confocal, two-photon and structured illumination fluorescence microscopy. Our findings suggest that the properties of symmetrical pentamethinium salts are affected to a higher extent by the type of a side unit rather than fluorine atoms on the γ-aryl moiety. Last but not least, we believe that such probes will further extend tools for mitochondrial research.