Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

Area of Science:

• Biophysics
• Optical Microscopy
• Materials Science

Background:

• Super-resolution fluorescence imaging relies on sequential activation and localization of fluorophores.
• The quality of super-resolution images is critically dependent on the properties of the fluorescent probes used.

Purpose of the Study:

• To quantitatively characterize the switching properties of 26 organic dyes.
• To directly relate probe properties to super-resolution image quality.
• To provide guidelines for probe selection and characterization.

Main Methods:

• Systematic characterization of 26 organic dyes, evaluating parameters like photon emission, duty cycle, photostability, and switching cycles.
• Direct correlation of these photophysical properties with the resulting super-resolution image quality.
• Demonstration of four-color super-resolution imaging with selected probes.

Main Results:

• Identified several photoswitchable organic dyes exhibiting good to excellent performance across four spectral ranges.
• Established a quantitative relationship between dye properties and super-resolution imaging outcomes.
• Successfully demonstrated low-cross-talk, four-color super-resolution imaging.

Conclusions:

• The study offers a valuable resource for selecting optimal fluorescent probes for super-resolution microscopy.
• The findings facilitate improved super-resolution image quality and enable advanced multi-color imaging applications.
• Provides a framework for systematic evaluation of novel photoswitchable probes.