Rhodamine-Derived Fluorescent Dye with Inherent Blinking Behavior for Super-Resolution Imaging

Area of Science:

• Biophysics
• Chemical Biology
• Microscopy

Background:

• Super-resolution microscopy overcomes the diffraction limit for cellular imaging.
• Single-molecule localization microscopy (SMLM) relies on sparse, stochastically fluorescing molecules.
• Existing SMLM probes often require specific activation or treatment.

Purpose of the Study:

• To develop a novel fluorescent dye for efficient SMLM.
• To characterize a rhodamine derivative with intrinsic pH-dependent blinking properties.
• To assess its utility in super-resolution imaging applications.

Main Methods:

• Synthesis and functionalization of a rhodamine derivative.
• Characterization of photophysical properties, including pH-dependent blinking kinetics.
• Demonstration of the dye's performance in SMLM without external activators.

Main Results:

• The rhodamine derivative exhibits spontaneous, pH-dependent switching between bright and dark states.
• At pH 7, less than 0.5% of molecules are fluorescent, enabling stochastic activation.
• Blinking occurs on second-to-minute timescales, suitable for SMLM.
• The dye is bright, easy to synthesize, and functionalize.

Conclusions:

• This novel rhodamine dye is a powerful, self-activating probe for SMLM.
• It simplifies super-resolution imaging workflows by eliminating sample treatment needs.
• The dye offers significant potential for advancing nanoscale biological imaging.