pH-sensitive perylene bisimide probes for live cell fluorescence lifetime imaging

Several new perylene bisimide (PBI) probes comprising oligo-guanidine conjugates and cationic hydrogel nanoparticle structures were designed for sensing intracellular pH in live cell fluorescence lifetime imaging microscopy (FLIM). Using adherent mammalian cells (2D) and neurosphere (3D) cell models, we evaluated their performance by confocal FLIM-TCSPC. The nanoparticle PBI probe showed stable pH calibration and lifetime changes from 4.7 to 3.7 ns between pH 4.4 and 8 attributed to photo-induced electron transfer (PET). The molecular oligo-guanidine probe showed fast cell penetration and bright staining, but its calibration is affected by the microenvironment being unreliable for quantitative FLIM. Thus, nanoparticle structures are preferred for the design of quantitative pH measurement by FLIM. High brightness and photostability, efficient staining of different cell types and positive optical response to acidification in fluorescence intensity and lifetime modalities are the advantages of the nanoparticle PBI probes compared to conventional pH probes such as BCECF (2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein). Other PBI derivatives with stronger PET can be developed for future high-resolution FLIM of intracellular pH.