Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart
Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart
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用于对跳动的斑马鱼胚胎心脏进行长期 3D 延时成像的混合光学门控
DOI：
10.1101/526830
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Taylor J
中科院分区：
文献类型：
--
作者：
Taylor J
使用最新的成像和计算技术，在跳动的心脏中的结构、细胞和亚细胞过程的三维荧光延时成像是越来越可实现的目标。然而，先前的方法具有显著的局限性。使用药物暂时停止心脏会破坏心脏的生理状态，并且使用超高帧速率进行荧光图像采集会导致光毒性细胞损伤。实时触发成像，同步到心脏周期中的特定阶段，可以计算“冻结”心脏，以获取3D延时成像所需的最少数量的荧光图像。然而，到目前为止，没有解决方案能够将锁相维持到心动周期中的相同点超过约一小时。我们的新的混合光学门控系统保持相位锁定长达24小时，获取同步的三维+时间视频堆栈的未受干扰的heartin活体内。这种方法使我们能够使用表达细胞特异性荧光团的转基因鱼系在胚胎斑马鱼心脏中观察详细的发育、结构、细胞和亚细胞过程，包括活细胞分裂和细胞命运追踪。我们表明，我们的方法不仅提供了高空间和时间分辨率的三维成像，而且还避免了光毒性损伤，替代方法会引起可测量的伤害。这为心脏在正常生理状态下跳动时提供了极好的细胞和亚细胞成像，并为进一步研究心脏和体内其他运动的细胞和亚细胞结构开辟了新的和令人兴奋的机会。
Three-dimensional fluorescence time-lapse imaging of structural, cellular and sub-cellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the heart’s physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally “freeze” the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heartin vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structuresin vivo.
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