Parameter optimization of NanoJ-SRRF for live-cell microtubule imaging

Super-Resolution Radial Fluctuation (SRRF) enables live-cell super-resolution imaging, but requires careful parameter selection. Here, we quantify the impact of NanoJ-SRRF parameters on microtubule imaging using FWHM and SQUIRREL-based error mapping. Ring radius proved most critical, with values >1.0 degrading resolution and fidelity. Radiality magnification and axes in ring had minimal impact. Advanced parameters revealed pitfalls: "remove positivity constraint" degraded resolution by 43%, while gradient weighting catastrophically reduced fidelity (RSP = 0.204 ± 0.116). Temporal Radiality Average (TRA) outperformed Temporal Radiality Auto-Correlations (TRAC), milimizing artifacts. This study establishes the first evidence-based guidelines for live-cell tubulin imaging: ring radius ≤1.0, TRA mode prioritization, and avoidance of gradient weighting. Integrating FWHM and SQUIRREL offers a robust opitimization framework for cytoskeletal dynamics.