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 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.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts.
Daan
D
Brinks
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts.
Aaron J
AJ
Klein
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts; Department of Physics, Harvard University, Cambridge, Massachusetts; Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts. Electronic address: cohen@chemistry.harvard.edu.
Adam E
AE
Cohen
D000200:Action Potentials; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D061848:Optical Imaging; D017785:Photons
D008565:Membrane Proteins