Figure 3. Staining quality affects ΔF and F_RLI differently.

A. Dura in the left half of the field was protected with a small piece of tissue paper during dura permeabilization and staining. Blue and red highlights indicate the selected areas for which signal was plotted in subsequent panels. B. Epileptiform spike amplitudes (ΔF_epileptiform) and resting light intensity (F_RLI) were measured over time, as in Figure 2, and plotted against each other. ΔF_epileptiform/F_RLI is nonlinear over the experiment. Furthermore, well-stained areas (blue) show different patterns of ΔF_epileptiform/F_RLI change compared to poorly-stained areas (red). C. The timecourse of epileptiform spike amplitude (ΔF_epileptiform) is plotted as in Figure 2B. Signal from poorly-stained areas (red) were smaller overall in amplitude, and showed somewhat faster “bleaching” kinetics within the exponential phase. D. Timecourse of resting light intensity (F_RLI) is displayed as in Figure 2C. F_RLI was smaller overall in the poorly-stained half. The exponential time constant of the poorly-stained area is much slower, and a large qualitative difference in bleaching kinetics is also evident. These results demonstrate that the staining quality can affect the relationship between ΔF and F_RLI, introducing hard-to-control biases when using the standard ΔF/F_RLI normalization.