Abstract
Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.
Competing Interest Statement
The authors declare the following competing financial interest(s): K.J. and L.W. are inventors of the patent Cell-permeable fluorogenic fluorophores which was filed by the Max Planck Society.
