Correction: Abdelfattah et al., "A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices"

Abstract

[This corrects the article on p. 2458 in vol. 36, PMID: 26911693.].

Figure 3.

Characterization of FlicR1. A, Image of HEK293 cells expressing FlicR1 under the CMV promoter. Scale bar, 10 μm. B, Fluorescence response (top) to a triangle wave in membrane potential (bottom) from −100 mV to +50 mV. Fluorescence trace (acquired at 10 Hz) is filtered using a 15 point moving-average low-pass filter. ΔF/F₀ is calculated as (F − F₀)/F₀, where F is the fluorescence at a particular time point and F₀ is initial fluorescence. C, FlicR1 fluorescence response (top) from a representative cell to a square wave in membrane potential (bottom) from −70 mV to +30 mV. D, E, FlicR1 (D) and ArcLight Q239 (E) fluorescence as a function of membrane voltage in a representative HEK293 cell. Fluorescence (F/F_min, where F is the fluorescence intensity at a particular membrane voltage and F_min is the minimum fluorescence intensity over the range of −100 mV to +50 mV) is the mean of three ramp cycles from −100 mV to +50 mV and back. Fluorescence is plotted starting at −25 mV, depolarizing to +50 mV, hyperpolarizing to −100 mV, and then returning back up to −25 mV, as marked by the arrows. Fluorescence showed little hysteresis between increasing and decreasing voltage ramps. F, FlicR1 fluorescence response to a 100 mV step potential in HEK293 cells. Solid line shows fluorescence response at 34°C. Dotted line shows fluorescence response at 22°C. G, ArcLight Q239 fluorescence response to a 100 mV step potential in HEK293 cells at 22°C. Note the different time axis compared with F. H, Magnification of the “on” and “off” portions of 22°C fluorescence traces from FlicR (red) and ArcLight (black). I, Normalized bleaching curves for FlicR1, ArcLight, and ASAP1 in HEK239 cells. J, Time constants for photobleaching of FlicR1, ASAP1, and ArcLight Q239 in HEK293 cells using continuous 10 W/cm² 561 nm light illumination for FlicR1 and continuous 10 W/cm² 488 nm light illumination for ASAP1 and ArcLight Q239. Fluorescence was captured every 500 ms. Time constants are based on single exponential fits. Error bars indicate SEM for FlicR1 (n = 5 cells), ASAP1 (n = 5 cells), and ArcLight Q239 (n = 4 cells). K, Spectral characterization of FlicR1 in vitro. Shown are absorbance (solid black line), excitation (dotted red line), and emission (solid red line) of FlicR1. Fluorescence imaging for voltage sensitivity measurements was performed at 10 Hz. Step responses were recorded at 2 kHz for FlicR1 and 1 kHz for ArcLight Q239. Illumination intensities were 10 W/cm².