Multiplex imaging of Rho family GTPase activities in living cells

Methods Mol Biol. 2012;827:215-34. doi: 10.1007/978-1-61779-442-1_15.


Here, we provide procedures for imaging the Rho GTPase biosensors in both single and multiplex acquisition modes. The multiplex approach enables the direct visualization of two biosensor readouts from a single living cell. Here, we take as an example a combination of the RhoA biosensor based on a CFP/YFP FRET modality and the Cdc42 biosensor based on organic dyes that change fluorescence as a function of the local solvent polarity. We list the required optical components as well as cellular manipulation techniques necessary to successfully image these two ratiometric biosensors in a single living cell.

Publication types

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

MeSH terms

  • Animals
  • Biosensing Techniques / instrumentation
  • Biosensing Techniques / methods*
  • Enzyme Activation
  • HEK293 Cells
  • Humans
  • Mice
  • Molecular Imaging / methods*
  • Staining and Labeling
  • Transduction, Genetic
  • cdc42 GTP-Binding Protein / metabolism
  • rho GTP-Binding Proteins / metabolism*


  • cdc42 GTP-Binding Protein
  • rho GTP-Binding Proteins