Comprehensive Characterization of an “off/on” Rhodol‐Based Lysosomal Tracker for Orthogonal Cellular Analysis by Confocal Imaging

Two florescent xanthene‐cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave‐assisted reactions. Both forms named “off” (nonfluorescent lactam) and “on” (strongly fluorescent ring‐opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X‐ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron‐withdrawing groups (EWGs) exhibit an equilibrium between non‐fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine‐type dyes, the ring‐opened forms of fluorescein‐ and rhodol‐cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3‐E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.