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 similar to 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.

Automated summary

Two highly fluorescent xanthene-cyanamide lysosomal trackers emitting at similar wavelengths were synthesized from fluorescein and rhodol methyl esters using microwave-assisted reactions. The on and off forms of these trackers were extensively characterized using various analytical techniques. Unlike rhodamine-type dyes, the ring-opened forms of fluorescein- and rhodol-cyanamides exhibited stability in protic solvents under circumneutral and basic pH conditions. Confocal imaging of osteoblastic cells using an orthogonal combination of fluorescent dyes allowed for the differentiation of various organelles and nuclei.