Multiplexed 3D FRET imaging in deep tissue of live embryos.
Multiplexed 3D FRET imaging in deep tissue of live embryos.
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DOI：
10.1038/srep13991
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发表时间：
2015-09-21
影响因子：
4.6
通讯作者：
Peng L
中科院分区：
文献类型：
--
作者：
Zhao M;Wan X;Li Y;Zhou W;Peng L
目前的深层组织显微镜技术主要局限于荧光团的强度图，这极大地限制了它们在研究体内生化过程中的应用。我们提出了一种深层组织多重功能成像方法，以高空间分辨率探测活胚胎中的多个福斯特共振能量转移（FRET）传感器。该方法利用多个激发激光器同时对荧光寿命进行 3D 成像。通过三通道强度和寿命图像的定量分析，我们证明可以在显微分辨率下同时监测表达双 FRET 传感器的活体胚胎的 Ca2+ 和 cAMP 水平。该方法与目前可用于探测各种细胞生化功能的各种 FRET 传感器兼容。它为对整个活体生物体中的复杂细胞电路进行成像打开了大门。
Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms.