Resolution assessment of super-resolution microscopy imaging: structural and technical dependencies for cell biology

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biophysics

Background:

• Super-resolution fluorescence microscopy (SRM) visualizes nanoscale cellular structures.
• Systematic evaluation of resolution assessment methods across diverse biological structures and SRM modalities is lacking.

Purpose of the Study:

• To comparatively assess three resolution metrics: Full Width at Half Maximum (FWHM), decorrelation analysis, and Fourier Ring Correlation (FRC).
• To evaluate these metrics across two SRM techniques (SRRF and STED) and diverse subcellular structures (microtubules, mitochondria, nuclear pore protein Nup98).
• To establish a framework for context-appropriate resolution assessment in cell biology.

Main Methods:

• Comparative assessment of FWHM, decorrelation analysis, and FRC.
• Application across Super-resolution Radial Fluctuation (SRRF) and Stimulated Emission Depletion (STED) microscopy.
• Utilized microtubules, mitochondria, and nuclear pore protein Nup98 in HeLa/U2OS cells.

Main Results:

• Decorrelation analysis provided robust resolution estimates across all structures and modalities, outperforming FWHM for dense structures like nuclear pore complexes.
• FWHM was suitable for discrete structures (microtubules, mitochondria) but not for densely packed features.
• NanoJ-SQUIRREL's FRC analysis quantified SRRF resolution, confirming minimal reconstruction artifacts (RSP > 0.90).
• STED achieved higher resolution than SRRF, but photobleaching limited FRC application.

Conclusions:

• Decorrelation analysis offers universal robustness for resolution assessment in SRM across various structures and techniques.
• FWHM is appropriate for discrete structures, while NanoJ-SQUIRREL provides standardized artifact validation and FRC quantification for computational super-resolution.
• This study establishes a framework for selecting appropriate resolution assessment methods based on structural features and technical principles in cell biology.