Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.
Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.
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DOI：
10.1002/jbio.201500258
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发表时间：
2016-04
影响因子：
2.8
通讯作者：
French PM
中科院分区：
文献类型：
--
作者：
Andrews N;Ramel MC;Kumar S;Alexandrov Y;Kelly DJ;Warren SC;Kerry L;Lockwood N;Frolov A;Frankel P;Bugeon L;McGinty J;Dallman MJ;French PM
荧光寿命成像（FLIM）结合光学投影断层扫描（OPT）有可能在完整的透明或接近透明的活生物体（如斑马鱼幼虫）中绘制空间和时间的Förster共振能量转移（FRET）读数，从而提供一种在其生理环境中可视化细胞信号传导过程的方法。在这里，FLIM OPT的第一个应用程序，以读出活的转基因斑马鱼幼虫使用基因表达的FRET生物传感器的生物功能的报告。细胞凋亡，或程序性细胞死亡，通过对被Caspase 3切割的FRET生物传感器的活性进行成像而在3D中绘制，Caspase 3是细胞凋亡的关键效应子。虽然细胞凋亡是发育过程中自然发生的过程，但它也可以通过多种方式触发，包括通过伽马射线照射。FLIM OPT在此显示，通过受精后24小时γ辐照后Caspase 3活化的变化，能够在活斑马鱼幼虫中随时间监测细胞凋亡。在照射后3.5小时观察到显著的细胞凋亡，主要在头部区域。
Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3‐D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.