Signals of di-8-ANEPPS at high temporal resolution. The stained cell, generating action potentials under field stimulation, was line-scanned in a 5 LIVE slit scanner with line-interleaved excitation for an effective ratiometric frequency of 8 kHz. (A) Images F₁₁(x,t) and F₂₂(x,t) (averages of four individual line scans) obtained with protocols illustrated in Fig. 1 and normalized (here and in the other panels of this figure) by the corresponding baseline averages F₁₁(x,0) and F₂₂(x,0). (B) The SEER ratio R(x,t) of the images in A and its line average R(t) (black trace). (C) Line-averaged fluorescence transients, F₁₁(t), F₁₂(t), F₂₁(t), and F₂₂(t), obtained at wavelengths detailed with Fig. 1, averaged over 10 action potentials and normalized to their initial value. (D) The three possible ratios that include F₂₂. Note that the SEER ratio (F₂₂/F₁₁) reaches the greatest amplitude. ID: 091912a_46 series 46.