Bright photoactivatable fluorophores for single-molecule imaging

Area of Science:

• Chemical Biology
• Microscopy
• Molecular Imaging

Background:

• Small-molecule fluorophores are essential for advanced live-cell imaging.
• Previous work introduced Janelia Fluor (JF) dyes, improving small, cell-permeable fluorophores.
• There is a need for photoactivatable variants for enhanced live-cell labeling strategies.

Purpose of the Study:

• To synthesize photoactivatable derivatives of existing Janelia Fluor (JF) dyes.
• To evaluate the compatibility of these new dyes with live-cell labeling.
• To assess the performance of activated dyes in advanced microscopy techniques.

Main Methods:

• Chemical synthesis of novel photoactivatable azetidine-containing fluorophores.
• Live-cell labeling experiments to assess compatibility and efficiency.
• Photophysical characterization including brightness and photostability measurements.
• Application in single-particle tracking and localization microscopy.

Main Results:

• Successful synthesis of photoactivatable Janelia Fluor (JF) dye derivatives.
• Demonstrated compatibility with live-cell labeling protocols.
• Activated dyes exhibit high brightness and excellent photostability, comparable to parent JF dyes.
• Enabled improved resolution and tracking in single-particle tracking and localization microscopy.

Conclusions:

• Photoactivatable Janelia Fluor (JF) dyes offer a powerful tool for advanced live-cell imaging.
• These derivatives maintain the superior properties of JF dyes after activation.
• The developed dyes facilitate high-performance single-particle tracking and localization microscopy, expanding their utility.