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. 2021 Mar 17;120(10):1927–1941. doi: 10.1016/j.bpj.2021.03.010

Figure 2.

Figure 2

Intermolecular FRET GEVIs. (A) Optical traces from ArcLight-CFP. HEK 293 cells expressing only ArcLight-CFP were voltage clamped at a holding potential of −70 mV and subjected to a five-pulse protocol (black trace). Excitation light for ArcLight-CFP was 430 nm. The optical trace in blue was acquired at 1 kHz. The solid blue line represents the mean from six cells. The shaded area is the standard error (SE) of the mean. (B) Optical traces from ArcLight-YFP. HEK 293 cells expressing only ArcLight-YFP were voltage clamped as described in (A). Excitation light was 500 nm for ArcLight-YFP. The solid red line represents the mean from four cells. (C) Optical traces from intermolecular FRET GEVIs. The GEVI schematics represent the three potential interactions upon cotransfection of HEK 293 cells with ArcLight-CFP and ArcLight-YFP. Excitation at 430 nm limits the contribution of the ArcLight-YFP/ArcLight-YFP interactions because the excitation peak of YFP is near 500 nm. Optical emissions at 480 and 540 nm were collected simultaneously with the use of an optical splitter (see Materials and methods). The blue trace is the 480 nm emission average from five cells voltage clamped as described in (A). The red trace is the 540 nm emission average.