Variation in temperature does not account for cell-to-cell variability of GxTX-594 kinetics. Temperature dependence of GxTX-594 labeling was assessed by holding the cell bath solution at either 27°C or 37°C and stepping the membrane voltage from −80 mV to 0 mV for a measurement of k_ΔF at both temperatures. (A) Representative traces of GxTX-594 fluorescence intensity response to voltage changes at 27°C (black) and 37°C (gray). Smooth lines are fits of a monoexponential function (Eq. 1): 27°C, k_ΔF = 2.47 × 10⁻² ± 0.39 × 10⁻² s⁻¹; 37°C, k_ΔF = 7.54 × 10⁻² ± 0.54 × 10⁻² s⁻¹. Background subtraction was performed as in Fig. 6 B. (B)k_ΔF at 27°C and 37°C. The rate of fluorescence change was significantly faster at higher temperatures (Mann–Whitney P = 0.0005). From geometric means (bars), a Q₁₀ of 3.8 was calculated between 27°C and 37°C. Each circle represents one cell, n = 7 both groups. ***, P < 0.0001.