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Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt

Abstract

This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy–based FRET techniques. By using the specific example of the FRET probe Akind (Akt indicator), which is a version of Akt modified such that FRET occurs when the probe is activated by phosphorylation, indicating Akt activation. The protocol provides a detailed step-by-step description of sample preparation, image acquisition and analysis, including control samples, image corrections and the generation of quantitative FRET/CFP ratio images for both sensitized emission and spectral imaging. The sample preparation takes 2 d, equipment setup takes 2–3 h and image acquisition and analysis take 6–8 h.

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Figure 1: Schematic diagrams depicting the three conditions that must be met for efficient FRET.
Figure 2: Schematic diagram of the intramolecular Akind FRET probe.
Figure 3: Acceptor photobleaching within regions of interest (ROI).
Figure 4: Crucial excitation and emission cross talk corrections.
Figure 5: Spectral imaging of autofluorescence and the CFP-Venus Akind probe.
Figure 6: FRET-FLIM analysis of cytosolic GFP and a FRET-positive GFP-mCherry probe, as well as the effects of the local fluorophore environment.
Figure 7: Calculation of FRET/CFP ratio images.
Figure 8: Comparison of the FRET ratio images for wild-type and mutant Akind probes.
Figure 9: Spectral imaging FRET of WT-Akind and 3A-Akind.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grant no. GM092914 to D.J.W. J.A.B. was supported by predoctoral training grant no. CA078136 from the NIH. B.R. was supported by a Swiss National Science Foundation fellowship (PA00P3_131496). The McGill Life Sciences Complex Imaging Facility was used for all microscopy and some of the data analysis for the work presented. The Imaging Facility is supported by the Canadian Foundation for Innovation and the Ministère du Développement Économique, innovation et exportation Québec. We thank J.-S. Lee for critical reading of the manuscript. Akind FRET probes were kindly provided by M. Matsuda (Kyoto University). Venus-C1 was a kind gift from A. Miyawaki (RIKEN, Japan).

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C.M.B. and J.A.B. developed and implemented the sensitized emission protocol, prepared the majority of the figures and drafted the manuscript. C.M.B. developed and implemented the spectral imaging protocol. J.A.B. performed all of the image analysis. D.J.W. contributed to the writing of the manuscript and provided substantial scientific feedback. B.R. participated in writing the manuscript, collected data and designed the FRET-FLIM introductory figures.

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Correspondence to Claire M Brown.

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Broussard, J., Rappaz, B., Webb, D. et al. Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt. Nat Protoc 8, 265–281 (2013). https://doi.org/10.1038/nprot.2012.147

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