SRRF (Super-Resolution Radial Fluctuation)-Based Nanoscale Imaging of Living Mitochondria: Key Parameters and Optimization Strategies
FANG Sanhua*, LIU Li, YANG Dan, LIU Shuangshuang, YIN Wei, ZHAO Qianbing, HUANG Qiong, CHEN Jingyao
As a computational imaging method requiring no specialized hardware, SRRF (super-resolution radial fluctuation) provides a powerful tool for live-cell super-resolution imaging, yet its performance is highly dependent on parameter settings. To establish an optimized SRRF imaging protocol for live-cell mitochondria, this study utilized HeLa cells as a model, labeled mitochondria with Abberior Live Orange Mito dye, acquired time series images via a spinning disk confocal microscope, and evaluated the effects of key parameters such as ring ra dius, radial magnification, and temporal analysis modes using the NanoJ-SRRF system. The resolution and quality of reconstructed images were quantitatively assessed using Full Width at Half Maximum and the resolution-scale Pearson correlation coefficient along with resolution-scale error from NanoJ-SQUIRREL. Results indicate that ring radius is the most critical parameter, with smaller values (0.1–1.0) achieving resolution beyond the diffraction limit; radial magnification and axes per ring have minor impacts on structural quality. Among temporal analysis modes, temporal radiality averaging provides the most stable and artifact-free reconstruction, whereas gradient weighting introduces significant artifacts and markedly reduces image quality. This study provides a quantitatively evaluated NanoJ-SRRF parameter optimization scheme under the experimental conditions of spinning disk confocal imaging, Abberior Live Orange Mito labeling, 108 nm pixel size, 20 Hz acquisition, and a 100-frame time series. It offers a reliable parameter starting point for nanoscale live-cell mitochondrial imaging under these specific conditions.
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