Extracellular access can impact GxTX-594 labeling kinetics. (A) Time-lapse Airy disk images of the glass-adhered surface of a voltage-clamped Kv2.1-CHO cell in 9 nM GxTX-594. Time index is in the upper left corner of each panel, and membrane potential is indicated in the upper right corner. Color progression for pseudocoloring of fluorescence intensity is shown in vertical bar on right. Scale bar, 10 μm. (B) Airy disk image of the glass-adhered surface of a voltage-clamped Kv2.1-CHO cell in 9 nM GxTX-594. Gray lines indicate boundaries of ROIs. ROIs 1, 2, and 3 are concentric circles, each with a respective diameter of 1.8, 4.9, and 9.1 μm. ROI 4 was hand-drawn to contain the apparent cell surface. In all cells analyzed, ROIs 1–3 were concentric circles of the same sizes, while ROI 4 varied based on cell shape. Scale bar, 10 μm. (C) Representative traces of GxTX-594 intensity response to voltage changes. Red lines are monoexponential fits (Eq. 1): 40-mV step ROI 1, k_ΔF = 4.29 × 10⁻² ± 0.26 × 10⁻² s⁻¹; ROI 2, k_ΔF = 4.39 × 10⁻² ± 0.16 × 10⁻² s⁻¹; ROI 3, k_ΔF = 5.65 × 10⁻² ± 0.15 × 10⁻² s⁻¹; and ROI 4, k_ΔF = 9.69 × 10⁻² ± 0.33 × 10⁻² s⁻¹. −80-mV step ROI 1, k_ΔF = 4.27 × 10⁻⁴ ± 0.11 × 10⁻⁴ s⁻¹; ROI 2, k_ΔF = 7.999 × 10⁻⁴ ± 0.053 × 10⁻⁴ s⁻¹; ROI 3, k_ΔF = 2.7556 × 10⁻³ ± 0.0074 × 10⁻³ s⁻¹; and ROI 4, k_ΔF = 5.46 × 10⁻³ ± 0.13 × 10⁻³ s⁻¹. Background for subtraction was the average intensity of a region that did not contain cells over the time course of the voltage protocol. Each trace was normalized to initial fluorescence intensity before the application of the voltage stimulus. (D)k_ΔF at +40 mV from individual cells. (E)k_ΔF at −80 mV from individual cells. Circle coloring in D and E indicates data from the same cell.