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Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors


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.

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Figure 1: The basic structure of a single-chain FRET-based biosensor.
Figure 2: Experimental flow.
Figure 3: Image acquisition.
Figure 4: Ratio image generation procedure.
Figure 5: Ratio imaging artifacts.
Figure 6: Basic controls used during FRET imaging setup.
Figure 7: Adaptation of the RacFRET biosensor for measurements in zebrafish embryos.
Figure 8: Controls used for measuring Rac activity.
Figure 9: Examples of FRET images in zebrafish.


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This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Max Planck Society. We thank M. Reichman-Fried for critical reading of the manuscript and E.-M. Messerschmidt for technical assistance. We also thank M. Matsuda and P. Daugherty for sharing the constructs.

Author information

Authors and Affiliations



E.K. designed and performed the experiments, analyzed the data and wrote the paper. J.B. designed and performed the experiments, analyzed the data and wrote the paper. E.R. supervised the project, designed the experiments and wrote the paper.

Corresponding author

Correspondence to Erez Raz.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Raw data captured in the YFP channel, of a cell expressing SECFP-YPet construct with the histogram showing the pixel intensity distribution. The maximum intensity value detected by the camera is 4095. To generate the histogram, the 16 bit image is first converted to 32 bit using the command: Image > Image Type > 32 bit from the ImageJ menu. To obtain the histogram, the command: Analyse > Histogram was used and the desired range was defined in the Histogram dialog box. (JPG 460 kb)

Supplementary Data 1

rawCFP image. The CFP emission channel of the somatic cell expressing RacFRET-noCT construct. (It is recommended that this file is viewed using ImageJ, a public domain Java image processing program available at (TIFF 51 kb)

Supplementary Data 2

rawYFP image. The YFP emission channel of the somatic cell expressing RacFRET-noCT construct. (It is recommended that this file is viewed using ImageJ, a public domain Java image processing program available at (TIFF 51 kb)

Supplementary Table 1

List of example DNA constructs used in this protocol (DOC 964 kb)

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Kardash, E., Bandemer, J. & Raz, E. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat Protoc 6, 1835–1846 (2011).

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