Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy

Area of Science:

• Chemical Biology
• Microscopy
• Organic Chemistry

Background:

• Rhodamines are crucial fluorophores for live-cell fluorescence microscopy.
• Their utility relies on a dynamic equilibrium between fluorescent zwitterions and nonfluorescent, cell-permeable spirocyclic forms.
• Tuning this equilibrium is challenging but essential for specific imaging applications.

Purpose of the Study:

• To develop a method for precisely controlling the rhodamine spirocyclization equilibrium.
• To create versatile rhodamine probes for diverse live-cell imaging techniques.
• To generate probes optimized for stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM).

Main Methods:

• Modification of the *ortho*-carboxy moiety of rhodamines into substituted acyl benzenesulfonamides and alkylamides.
• Systematic tuning of the spirocyclization equilibrium over a wide range with high accuracy.
• Synthesis of differently colored probes for various labeling systems and imaging needs.

Main Results:

• Demonstrated precise control over the rhodamine equilibrium through chemical modification.
• Successfully transformed rhodamines into highly fluorogenic, cell-permeable probes for live-cell STED microscopy.
• Developed spontaneously blinking rhodamine dyes suitable for single-molecule localization microscopy (SMLM).

Conclusions:

• The described chemical modification strategy offers unprecedented accuracy in tuning rhodamine photophysical properties.
• This approach enables the creation of tailored rhodamine probes for advanced live-cell imaging applications.
• The generated probes are versatile, supporting both STED and SMLM with optimized performance.