Biophysical Society Thematic Meeting - June 28-July 1, 2015

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New Biological Frontiers Illuminated by Molecular Sensors and Actuators Poster Abstracts
1-POS Board 1
Expanding the Toolbox of Genetically Encoded Voltage Indicators
Ahmed S. Abdelfattah1, Samouil L. Farhi2 , Yongxin Zhao1, Daan Brinks2, Adam E. Cohen2,
Robert E. Campbell1.
1University of Alberta, Edmonton, AB, Canada, 2Harvard University, Cambridge, MA, USA.
Simultaneous recording of neuronal activity from many locations is key to understanding how
brain function emerges from neuronal circuits. Optical imaging using voltage indicators based on
green fluorescent proteins (FPs) has emerged as a powerful approach for detecting the activity of
many individual neurons with high spatial and temporal resolution. To expand the toolbox of
genetically encoded voltage indicators, we engineered two FP-based voltage indicators: A bright
red fluorescent voltage indicator (FlicR1) and a green to red highlightable voltage indicator
(FlicGR). We used directed protein evolution to screen libraries of thousands of variants to
identify clones with sufficient brightness and response amplitude that would report membrane
potential changes in mammalian cells.
FlicR1 has voltage sensing properties that are comparable to the best available green indicators.
It reports single action potentials in neurons in single trial recordings. Furthermore, FlicR1 can
be imaged with wide field fluorescence microscopy using a typical mercury arc lamp, and
faithfully reports spontaneous activity in cultured hippocampal neurons and rat brain organotypic
slices. We also demonstrate that FlicR1 can be used in conjunction with PsChR, a blue-shifted
channelrhodopsin, for all-optical neuronal activation and activity recording, although blue light
photoactivation of the FlicR1 chromophore remains a challenge.
FlicGR is the first example of a highlightable voltage indicator. A powerful application of
fluorescent proteins is their use as highlighters where they can be converted from one color to
another by light. Using blue light, we can photoconvert FlicGR from green to red. We
demonstrate that both the green and red forms are sensitive to membrane potential changes in
mammalian cells.