Fig. 5.

Image analysis and intensity dependence of Ca²⁺ free rises in dissociated ommatidia.

(A) Six frames (0–2s) from a 100 Hz (10 ms exposure) movie (see Movie 2) of wild-type ninaE-GCaMP6f ommatidium in Ca²⁺ free bath (0 Ca²⁺, 1 mM EGTA 120 mM Na⁺). Brightness and contrast in all frames auto-adjusted to the final (brightest) frame. Bright spots towards distal (right) end of the ommatidium are autofluorescent pigment granules. Scale bar 10 μm. (B) Average time-courses (n = 6 ommatidia) from regions of interest covering rhabdomeres (rh) and cytosol (cyt) show near perfect overlap. Traces normalised to facilitate comparison of time-course: maximum ΔF/F₀ values were in range 2–5. (C) Rising phase on faster time base. (D) PMT fluorescence traces from a wild-type ninaE-GCaMP6f ommatidium, perfused with Ca²⁺ free bath (1 mM EGTA) for ∼30s. Brief (10 ms) green flashes of increasing intensity (0, 4000, 20000, 70000 and 600000 effective photons) were presented 2 s before the 2 s blue excitation. The instantaneous fluorescence “pedestals” (arrows) reflects the Ca²⁺ level reached in response to the green flashes. (D) Resulting F/log I function (mean ± S.E.M. n = 4) compared to data from ommatidia in normal bath (1.5 mM Ca²⁺ replotted from Fig. 3).