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

• 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
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2015.07.038

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.