posted on 2021-08-30, 20:31authored byNicolas Lardon, Lu Wang, Aline Tschanz, Philipp Hoess, Mai Tran, Elisa D’Este, Jonas Ries, Kai Johnsson
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 nonfluorescent but cell-permeable spirocyclic form. Different imaging
applications require different positions of this dynamic equilibrium,
and an adjustment of the equilibrium 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 one to transform the same rhodamine
into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated
emission depletion (STED) microscopy or 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.