Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage

Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage...

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Bibliographic Details
Published in:Biophysical journal 2015-09, Vol.109 (5), p.914-921
Main Authors: Brinks, Daan, Klein, Aaron J., Cohen, Adam E.
Format: Article
Language:English
Subjects:
Action Potentials
Atoms & subatomic particles
Cell Biophysics
Cells
Electric currents
Fluorescence
HEK293 Cells
Humans
Membrane Potentials
Membrane Proteins - metabolism
Membranes
Optical Imaging
Photons
Proteins
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Summary:Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2015.07.038