title: Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.
abstract: Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.
authors.affiliation: Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University; Center for Functional Connectomics, Korea Institute of Science and Technology.
authors.forename: Sungmoo
authors.initials: S
authors.lastname: Lee
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology.
authors.forename: Hong Hua
authors.initials: HH
authors.lastname: Piao
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology.
authors.forename: Masoud
authors.initials: M
authors.lastname: Sepheri-Rad
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology; College of Life Sciences and Biotechnology, Korea University.
authors.forename: Arong
authors.initials: A
authors.lastname: Jung
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology.
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.affiliation: Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University; Advanced Institutes of Convergence Technology.
authors.forename: Yoon-Kyu
authors.initials: YK
authors.lastname: Song
authors.affiliation: Center for Functional Connectomics, Korea Institute of Science and Technology; bradley.baker19@gmail.com.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baker
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008564:Membrane Potentials / Q000502:physiology*; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins