Optimizing the resolution-fidelity trade-off in SRRF nanoscopy for live-cell clathrin imaging

Area of Science:

• Cell biology
• Microscopy
• Nanotechnology

Background:

• Live-cell imaging of clathrin-coated pits (CCPs) is essential for understanding endocytosis.
• Total internal reflection fluorescence microscopy (TIRFM) is diffraction-limited, hindering visualization of CCP nanostructures.
• Super-Resolution Radial Fluctuations (SRRF) offers computational super-resolution but requires careful parameter optimization.

Purpose of the Study:

• To establish a comprehensive parameter optimization framework for SRRF-based nanoscopy of live CCPs.
• To resolve the trade-off between resolution and structural fidelity in SRRF imaging.
• To enable robust nanoscale imaging of clathrin-mediated endocytosis.

Main Methods:

• Utilized multi-modal metrics in CLTA-GFP HeLa cells for parameter optimization.
• Evaluated different SRRF modes, including Temporal radiality pairwise product mean (TRPPM), Temporal radiality average (TRA), and temporal radiality auto-correlations (TRAC).
• Assessed parameters like ring radius, 'remove positivity constraint', and gradient weighting.

Main Results:

• Identified an optimal ring radius of 1.0, balancing resolution (180 nm) and fidelity (Pearson = 0.935).
• TRPPM mode achieved superior resolution (154 nm) while maintaining high fidelity.
• Parameters 'remove positivity constraint' and gradient weighting induced artifacts and were detrimental.

Conclusions:

• The optimized SRRF framework significantly improves nanoscale imaging of live CCPs.
• This approach overcomes the resolution-fidelity limitations of conventional TIRFM.
• Enables detailed visualization of clathrin-mediated endocytosis dynamics at the nanoscale.