Kolossov Vladimir L, Sivaguru Mayandi, Huff Joseph, Luby Katherine, Kanakaraju Kaviamuthan, Gaskins H Rex
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Microscopy and Imaging Core Facility, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Microsc Res Tech. 2018 Feb;81(2):115-128. doi: 10.1002/jemt.22968. Epub 2017 Nov 13.
Mitochondrial morphology is regulated by continuous fusion-and-fission events that are essential for maintaining normal function. Despite the prominence of mitochondrial function in energy generation and cell signaling, understanding of processes of fusion and fission dynamics has been hampered by the lack of high-resolution optical systems that accommodate live-cell imaging. We have examined different confocal modalities in terms of resolution and signal-to-noise ratio (SNR) in a point scanning confocal microscope with Airyscan super-resolution (AS-SR). Results indicated that Airyscan (AS) provided speed, super-resolution, and high SNR. This modality was then used for monitoring mitochondrial dynamics in live tumor cells modified to harbor green-fluorescent protein localized to mitochondria. We then compared regular AS and fast-Airyscan modalities in terms of gentleness on the live-cell samples. The fast mode provided unprecedented imaging speed that permits monitoring dynamics both in 2D and also in three-dimensional dataset with time lapses (4D). Alterations to the mitochondrial network in U87 glioblastoma cells occurred within seconds and the cells were not affected by modest inhibition of fission. The super-resolution permitted quantitative measurements of mitochondrial diameter with a precision that enabled detection of significant differences in mitochondrial morphology between cell lines. We have observed swelling of mitochondrial tubules in A549 lung cancer cells after 2 hr treatment with deoxynyboquinone, an ROS-generating pharmacologic drug. We also tested different 3D analytical parameters and how they can affect morphometric quantitation. The AS-SR imaging enabled high-speed imaging of mitochondrial dynamics without the compromise to cell morphology or viability that is common with conventional fluorescence imaging due to photo-oxidation.
线粒体形态由持续的融合和分裂事件调控，这些事件对于维持正常功能至关重要。尽管线粒体功能在能量产生和细胞信号传导中十分突出，但由于缺乏适用于活细胞成像的高分辨率光学系统，对融合和分裂动力学过程的理解受到了阻碍。我们在配备Airyscan超分辨率（AS-SR）的点扫描共聚焦显微镜中，从分辨率和信噪比（SNR）方面研究了不同的共聚焦模式。结果表明，Airyscan（AS）提供了速度、超分辨率和高信噪比。然后，这种模式被用于监测经改造以携带定位于线粒体的绿色荧光蛋白的活肿瘤细胞中的线粒体动力学。接着，我们在对活细胞样本的温和性方面比较了常规AS模式和快速Airyscan模式。快速模式提供了前所未有的成像速度，允许在二维以及带有时间推移的三维数据集中（4D）监测动力学。U87胶质母细胞瘤细胞中线粒体网络的变化在数秒内发生，并且细胞不受适度的裂变抑制影响。超分辨率允许对线粒体直径进行定量测量，其精度能够检测细胞系之间线粒体形态的显著差异。我们观察到，在用产生ROS的药物脱氧尼博醌处理2小时后，A549肺癌细胞中的线粒体小管出现肿胀。我们还测试了不同的三维分析参数以及它们如何影响形态计量学定量。AS-SR成像能够对线粒体动力学进行高速成像，而不会像传统荧光成像因光氧化那样对细胞形态或活力造成损害。
