Alternative ratiometric signals. (A) Line scan image of fluorescence F₂₂(x,t), corrected for bleaching and staining inhomogeneity, then cropped and resized for convenience of presentation, acquired in emission range Em2 upon excitation with Ex1 (Fig. 1 B). While the images were being acquired, a train of 15 field stimuli was applied at a frequency of 40 Hz. (B) The spatial average F₂₂(t) of F₂₂(x,t). (C) The spatially averaged emission ratio, of F₂₂(t) by the corresponding average of F₂₁(x,t), acquired simultaneously in emission range Em1. (D) The averaged excitation ratio, of F₂₂(t) by the average of F₁₂(x,t), acquired in the same emission range while excited by Ex1. (E) The SEER ratio, F₂₂(t)/F₁₁(t), which produces the greatest signals among all possible combinations. The ordinate scales of graphs B–D are chosen to make the span of the transients approximately proportional to the relative change in ratio. ID: 030612a_049.