New fluorinated rhodamines for optical microscopy and nanoscopy

Area of Science:

• Organic Chemistry
• Biochemistry
• Microscopy

Background:

• Rhodamine dyes are widely used as fluorescent markers.
• Existing rhodamine dyes can suffer from photobleaching and limited suitability for high-intensity imaging.
• Development of novel, photostable fluorescent probes is crucial for advanced microscopy.

Purpose of the Study:

• To synthesize and characterize new photostable rhodamine dyes with unique structural modifications.
• To evaluate the photophysical properties and performance of these novel dyes in various microscopy applications.
• To explore their potential as fluorescent labels for biomolecules and live-cell imaging.

Main Methods:

• Synthesis of novel rhodamine derivatives (compounds 1a-r, 3-5) with fluorinated and sulfonated xanthene fragments.
• Characterization of photophysical properties including excitation/emission wavelengths, quantum yields, and excited-state lifetimes.
• Assessment of photostability under high laser intensities relevant to confocal microscopy.
• Evaluation of compatibility with bioconjugation strategies via a secondary amide bond.
• Demonstration of performance in fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy.
• Testing of cell permeability for live-cell labeling applications.

Main Results:

• Novel rhodamine dyes with N',N-bis(2,2,2-trifluoroethyl) groups and fluorinated xanthene fragments were synthesized.
• Compounds exhibit high fluorescence quantum yields (up to 98%) and long excited-state lifetimes (>3 ns).
• Dyes demonstrate exceptional photostability, particularly under high laser intensities.
• Sulfonation is compatible with conjugation-ready carboxylic acid groups for biomolecule labeling.
• Fluorinated rhodamines successfully permeated the plasma membrane of living cells.
• Excellent performance was shown in FCS and STED nanoscopy experiments.

Conclusions:

• The newly developed rhodamine dyes offer a unique combination of photostability, high fluorescence, and cell permeability.
• These dyes are highly suitable for demanding optical microscopy and nanoscopy techniques, including live-cell imaging.
• The structural modifications enable versatile bioconjugation, expanding their utility as fluorescent labels.