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 Orang...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。展开更多
We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,...We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.展开更多
文摘超分辨率径向波动(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。
基金supported by the Ministry of Science,ICT and Future Planning(MSIP)through the National Research Foundation of Korea(NRF)(RS-2024-00450201)supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute(KHIDI),funded by the Ministry of Health and Welfare,Republic of Korea(HI22C1374).
文摘We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.
