Voltage Imaging with ANNINE Dyes and Two-Photon Microscopy

  • Christopher J. Roome
  • Bernd KuhnEmail author
Part of the Neuromethods book series (NM, volume 148)


Voltage imaging is a tried and tested tool for revealing changes in neuronal membrane voltage at high temporal and spatial resolution in vitro and in vivo. However, single-photon in vivo voltage imaging using cameras and synthetic dyes does not allow depth resolution in highly scattering tissue such as the mammalian brain and risks introducing artifacts due to phototoxicity and bleaching. In contrast, voltage imaging with synthetic electrochromic dyes and two-photon excitation near the red spectral edge of absorption circumvents these challenges, allowing depth-resolved measurement of voltage changes at high spatial and temporal resolution in scattering tissue with negligible phototoxicity and bleaching.

Here, we describe how to image voltage using two-photon microscopy and the voltage-sensitive dyes ANNINE-6 and ANNINE-6plus. The key advantages of these dyes are that the voltage response is linear, the temporal resolution is essentially limited by the imaging speed, and phototoxicity and bleaching can be neglected when an excitation wavelength close to the red spectral edge of absorption is chosen. We report how to image membrane voltage of dissociated cells in culture. We provide protocols for imaging average membrane voltage in vitro (in brain slices) and in anesthetized and awake animals. Finally, we describe the labeling of single neurons in vivo and how to measure supra- and sub-threshold voltage changes in their dendrites in the awake animal. Voltage can be imaged from internally labeled cells for at least 2 weeks after a single electroporation. Dendritic voltage imaging can be combined with electrical recording, calcium imaging, and/or pharmacology.


Voltage-sensitive dye VSD Voltage imaging ANNINE Membrane potential Two-photon In vitro In vivo 



The authors thank Espen Hartveit, Steven D. Aird, Neil Dalphin, Ray X. Lee, Mohamed M. Eltabbal, Alisher Duspayev, Claudia Cecchetto, and Leonidas Georgiou for valuable feedback on the manuscript and the Okinawa Institute of Science and Technology Graduate University for internal funding.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Okinawa Institute of Science and Technology Graduate UniversityOkinawaJapan

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