Chapter 20 Genetically Encoded Protein Sensors of Membrane Potential Douglas Storace, Masoud Sepehri Rad, Zhou Han, Lei Jin, Lawrence B. Cohen, Thom Hughes, Bradley J. Baker, and Uhna Sung Abstract Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. However these dyes suffer from the drawbacks of non-specificity of cell staining and low accessibility of the dye to some cell types. Further progress in imaging activity is expected from the devel- opment of genetically encoded fluorescent sensors of membrane potential. Cell type specificity of expression of these fluorescent protein (FP) voltage sensors can be obtained via several different mechanisms. One is cell type specificity of infection by individual virus subtypes. A second mechanism is specificity of promoter expression in individual cell types. A third, depends on the offspring of transgenic animals with cell type specific expression of cre recombinase mated with an animal that has the DNA for the FP voltage sensor in all of its cells but its expression is dependent on the recombinase activity. Challenges remain. First, the response time constants of many of the new FP voltage sensors are slower (2–10 ms) than those of organic dyes. This results in a relatively small fractional fluorescence change, ΔF/F, for action potentials. Second, the largest signal presently available is only ~40 % for a 100 mV depolarization and many of the new probes have signals that are substantially smaller. Large signals are especially important when attempting to D. Storace • Z. Han • L. Jin Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA M.S. Rad • B.J. Baker • U. Sung Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 136-791, Korea L.B. Cohen (*) Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 136-791, Korea Department of Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510, USA e-mail: lawrence.b.cohen@hotmail.com T. Hughes Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717, USA © Springer International Publishing Switzerland 2015 M. Canepari et al. (eds.), Membrane Potential Imaging in the Nervous System and Heart, Advances in Experimental Medicine and Biology 859, DOI 10.1007/978-3-319-17641-3_20 493