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. 2021 Jul 22;10:e68955. doi: 10.7554/eLife.68955

Figure 1. Schematic of dipicrylamine (DPA) quenching measurements.

(A) Absorption (dotted line) or fluorescence emission (solid lines) spectra of DPA (black), CFP variant mTurqoise2 (blue), GFP variant Gamillus (green) and YFP variant mVenus (yellow). Structure of DPA inset. (B, left) DPA (orange circle) localizes to the outer leaflet with hyperpolarization and moves to inner leaflet upon depolarization, resulting in stronger quenching. Arrows reflect excitation (purple), emission (blue), or resonance transfer (black). (B, right) Exemplar patch fluorometry data of Lck-CFP fluorescence changing during voltage steps. (C) Single cell fluorescence, normalized to fluorescence at −15 mV, over a range of voltages for Lck-CFP (blue) and Lck-YFP (yellow). Solid lines are fits to Boltzmann function which yield ΔF/F. (D) Summary of ΔF/Fs from Lck-CFP or Lck-YFP. Squares are single cells (N = 6 cells per construct) and error bars depict SEM.

Figure 1.

Figure 1—figure supplement 1. Non-linearity of fluorescence resonance energy transfer (FRET) provides for distance estimates.

Figure 1—figure supplement 1.

Simulated FRET efficiency over distance with an R0 of 55 Å. Moving donor and acceptor apart by a fixed distance has a larger effect when the donor and acceptor are close (donor proximal case) compared to when they are further apart (donor distal case).