title: 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.
authors.affiliation: Department of Biological Sciences and Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Broussard
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rappaz
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Webb
authors.forename: Claire M
authors.initials: CM
authors.lastname: Brown
mesh_terms: D004789:Enzyme Activation / Q000502:physiology*; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods; D038761:Photobleaching; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*
chemical_list: D051057:Proto-Oncogene Proteins c-akt