Characterization of voltage-sensitive dyes in living cells using two-photon excitation

Methods Mol Biol. 2013;995:147-60. doi: 10.1007/978-1-62703-345-9_11.


In this protocol, we describe the procedures we have developed to optimize the performance of voltage-sensitive dyes for recording changes in neuronal electrical activity. We emphasize our experience in finding the best dye conditions for recording backpropagating action potentials from individual dendritic spines in a neuron within a brain slice. We fully describe procedures for loading the dye through a patch pipette and for finding excitation and emission wavelengths for the best sensitivity of the fluorescence signal to membrane voltage. Many of these approaches can be adapted to in vivo preparations and to experiments on mapping brain activity via optical recording.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Action Potentials
  • Animals
  • Dendritic Spines / physiology
  • Fluorescence
  • Fluorescent Dyes / chemistry*
  • Humans
  • Sensitivity and Specificity
  • Signal-To-Noise Ratio
  • Single-Cell Analysis / methods
  • Software
  • Styrenes / chemistry
  • Voltage-Sensitive Dye Imaging / methods*


  • Fluorescent Dyes
  • JPW 1114
  • JPW 3028
  • Styrenes