Figure 1. Fluorescence measured from a dye film.

Fluorescence measured from a gel film approximately 130 μm thick containing 50 μm free fluo-5F in Ringer solution with 0.5 % Agar (see Methods). A, continuous 60 s exposure of the dye film to the laser spot at an intensity of 2.2 × 10⁹ photons μm⁻² s⁻¹, similar to that used for measurements with continuous laser illumination from rod outer segments (see e.g. Fig. 2A). Trace is average of 8 measurements. Timing of laser illumination is indicated by upper trace. Inset, same data at higher temporal resolution. B, exposure of same dye film to 6 laser flashes 100 ms in duration repeated at 5 s intervals. Intensity of laser was 2.2 × 10¹⁰ photons μm⁻² s⁻¹, similar to that used for this laser flash protocol on rod outer segments (see e.g. Fig. 3A). Trace is average of 4 measurements. Amplitudes of fluorescence responses were within 1-2 % of one another and showed no evidence of a systematic decrease indicative of dye bleaching. Timing of laser illumination is indicated by upper trace.