H-Rubies, a new family of red emitting fluorescent pH sensors for living cells

Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The widespread development of fluorescent imaging techniques makes pH sensitive fluorescent dyes valuable tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine fluorophore that display a bright red fluorescence upon acidification with pK(a) values spanning from 4 to 9. Slight structural modifications led to dramatic changes in their physicochemical properties and a relationship between their structures, their ability to form H-aggregates, and their apparent pK(a) was established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) using flow cytometry.