Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors
Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors
复制标题
DOI：
10.1038/nprot.2011.395
复制
发表时间：
2011-12-01
期刊：
影响因子：
14.8
通讯作者：
Raz, Erez
中科院分区：
文献类型：
--
作者：
Kardash, Elena;Bandemer, Jan;Raz, Erez
基于荧光共振能量转移（FRET）的分子生物传感器是研究活细胞中蛋白质活性的重要工具，在过去十年中已被广泛用于此目的。然而，由于活生物体本身具有高度的细胞复杂性以及与三维环境相关的成像难题，FRET生物传感器很少在活生物体环境中使用。在此，我们提供了一个在早期发育过程中使用基于单链分子内FRET的生物传感器的实验方案。我们提供了一个在胚胎中进行FRET比率成像的通用方案，包括数据采集条件和比率图像生成算法。然后，我们使用pRaichu RacFRET生物传感器来举例说明特定生物传感器在活斑马鱼胚胎中使用的调整和优化。一旦有了优化的生物传感器，包括将探针引入胚胎、成像和数据分析在内的整个过程需要2 - 3天。
Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2-3 d.