Chemistry

Materials science

Fluorescence

Artificial intelligence

Super-resolution microscopy

Physics

Fluorescence microscope

Nanotechnology

Organic chemistry

Optics

Fluorophore

Rhodamine

Moiety

Molecule

Computer science

Resolution (logic)

Microscopy

Zwitterion

Stereochemistry

Journal of the American Chemical Society

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.

Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy