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

Nat Protoc. 2013 Feb;8(2):265-81. doi: 10.1038/nprot.2012.147. Epub 2013 Jan 10.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Enzyme Activation / physiology*
  • Fluorescence Polarization
  • Fluorescence Resonance Energy Transfer / methods*
  • Image Processing, Computer-Assisted / methods*
  • Microscopy / methods*
  • Models, Molecular
  • Optical Imaging / methods
  • Photobleaching
  • Proto-Oncogene Proteins c-akt / metabolism*


  • Proto-Oncogene Proteins c-akt