【摘要】 超分辨率径向波动(SRRF)作为一种无需特殊硬件的计算成像方法,为活细胞超分辨率成像提供了有力工具,然而该技术的成像效果高度依赖参数设置。为建立针对活细胞线粒体的SRRF成像优化方案,该研究以HeLa细胞为模型,利用Abberior Live Orange Mito染料标记线粒体,通过转盘共聚焦显微镜获取时间序列图像,并使用NanoJ-SRRF系统评估了环半径、径向放大倍数、时序分析模式等关键参数对成像质量的影响。重建图像的分辨率与质量分别采用半高全宽和NanoJ-SQUIRREL的分辨率尺度皮尔逊相关系数与分辨率尺度误差进行定量评估。结果表明,环半径是最关键参数,较小取值(0.1～1.0)可实现超越衍射极限的分辨率;径向放大倍数与环内轴数对结构质量影响较小;在时序分析模式中,时序径向性平均法能提供最稳定且无伪影的重建结果;而梯度加权会引入明显伪影并显著降低图像质量。该研究针对转盘共聚焦成像、Abberior Live Orange Mito标记、108 nm像素尺寸、20 Hz采集、100帧时间序列的实验条件,提供了经量化评估的NanoJ-SRRF参数优化方案,为该条件下的活细胞线粒体纳米级成像提供了经量化评估的参数优化起点。更多还原

【Abstract】 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 timeseries images via a spinning disk confocal microscope, and evaluated the effects of key parameters such as ring radius, 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.更多还原