Lee, Sungmoo Piao, Hong hua Sepheri-Rad, Masoud Jung, Arong Sung, Uhna Song, Yoon-Kyu Baker, Bradley J. 2024-01-20T05:02:22Z 2024-01-20T05:02:22Z 2021-09-05 2016-02 1940-087X https://pubs.kist.re.kr/handle/201004/124448 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 Forster 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. English MYJoVE Corporation Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors Article 10.3791/53566 1 Journal of Visualized Experiments, v.-, no.108, pp.1 - 10 Journal of Visualized Experiments - 108 1 10 N scie scopus 000372504100037 2-s2.0-84959378536 Multidisciplinary Sciences Science & Technology - Other Topics Article ELECTRICAL-ACTIVITY OPTICAL ELECTROPHYSIOLOGY NEURONS BRIGHT Neuroscience Issue 108 Genetically encoded voltage indicator voltage imaging FRET image splitter fluorescent protein