Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Red-emitting fluorescent markers are crucial for minimizing cellular autofluorescence in biological microscopy.
• Enhanced flexibility in multicolor experiments is achieved with red emitters.
• Existing red fluorophores may have limitations in photostability or quantum yield.

Purpose of the Study:

• To develop novel rhodamine dyes with emission around 660 nm, excitable by 630 nm laser light.
• To synthesize lipophilic and hydrophilic derivatives for diverse biological applications.
• To evaluate the performance of these new dyes in various advanced microscopy techniques.

Main Methods:

• Synthesis of rhodamine derivatives with tunable solubility (lipophilic/hydrophilic) and reactive groups.
• Characterization of photophysical properties including quantum yield, excited state lifetime, and intersystem-crossing rates.
• Application of the dyes in conventional microscopy, super-resolution microscopy (STED, GSDIM), and single-molecule spectroscopy (FCS).

Main Results:

• Developed novel rhodamine dyes with high photostability, quantum yields up to 80%, and long excited state lifetimes (3.4 ns).
• Demonstrated excellent performance in STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return) microscopy.
• Showcased utility in single-molecule fluorescence correlation spectroscopy (FCS) and as tags in mass spectrometry (MS) and liquid chromatography-mass spectrometry (LC-MS).

Conclusions:

• The novel rhodamine dyes are highly versatile probes for advanced fluorescence microscopy and nanoscopy.
• Derivatives with sulfo groups exhibit enhanced water solubility and quantum yield.
• Deuterated dyes show potential as tags for sensitive identification and quantification in complex mixtures.