Abstract
We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd′) ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd′s = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca2+ in layers 8–10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower Kd′ was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.
Highlights
The procedure for purification and determination of extinction coefficient, quantum yield, and Kd′ of Y-GECO variants has been previously described[11]
Despite the fact that 30% of neurons in humans and Drosophila are inhibitory, there are relatively few optogenetic indicators optimized for imaging of inhibitory neuronal activity
Inhibitory activity could be more detected with an inverse response Ca2+ indicator due to the diminished background fluorescence originating from adjacent out-of-focus cells
Summary
The procedure for purification and determination of extinction coefficient, quantum yield, and Kd′ of Y-GECO variants has been previously described[11]. A DU-800 UV-vis spectrophotometer (Beckman) was used to measure absorption spectra, and a Safire[2] plate reader (Tecan) was used to measure the excitation and emission spectra. The ratiometric response to Ca2+ of Y-GECO is defined as (Rmax − Rmin)/Rmin, where R = (I with 526 nm excitation)/(I with 416 nm excitation), and I is the fluorescence intensity at 550 nm. The intensiometric response to Ca2+ of Y-GECO is defined as (Imax − Imin)/Imin, where I is the fluorescence intensity at 550 nm with 526 nm excitat
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