Lee Sungmoo, Piao Hong Hua, Sepheri-Rad Masoud, Jung Arong, Sung Uhna, Song Yoon-Kyu, Baker Bradley J
Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University; Center for Functional Connectomics, Korea Institute of Science and Technology.
Center for Functional Connectomics, Korea Institute of Science and Technology.
J Vis Exp. 2016 Feb 4(108):e53566. doi: 10.3791/53566.
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.
基因编码电压指示剂（GEVIs）已经发展到开始对体内记录有用的程度。虽然最终目标是对体内神经元活动进行成像，但必须能够对单个细胞的活动进行成像，以确保体内实验准备的成功。本实验步骤将描述如何对单个细胞的膜电位进行成像，为最终的体内成像提供基础。在这里，我们描述了在体外对由与单个荧光蛋白（FP）或两个能够进行荧光共振能量转移（FRET）的荧光蛋白融合的电压感应域组成的GEVIs进行成像的方法。使用图像分离器能够将由两个不同波长产生的图像同时投射到同一个电荷耦合器件（CCD）相机上。图像分离器在光路中放置了第二个滤光片组。这个第二个滤光片组由一个二向色镜和两个发射滤光片组成，根据GEVI的荧光蛋白来分离供体和受体荧光波长。这种设置能够在通过全细胞膜片钳配置操纵膜电位时，同时记录受体和供体荧光伴侣。当使用由单个FP组成的GEVI时，可以移除第二个滤光片组，使图像分离器中的镜子将单个图像投射到CCD相机上。
