Fig. 8

Responses of ArcLight to ITMV and additional illumination by different wavelengths of light. Top – induced transmembrane voltage (ITMV): Electrodes are positioned at the black arrows to the left of the MiCAM image. Root regions close to the electrodes that were used to make the graph are circled in red and blue to correspond to the cognate traces in the graph. Images were acquired at 200 ms intervals over a time period of 205 s. Voltage pulses (2.5 V with a duration of 200 ms) were applied at approximately 60 s and 120 s for normal (N) and reverse (R) polarities, respectively. ArcLight in the two regions responds in an opposite manner depending on the polarity of the pulse. The different effect in the two regions can be explained by the proximity of the responding cells to the depolarising electrode (i.e. cathode). With ‘normal polarity’ (stimulus at t = 60 s) the bottom electrode is the cathode and the blue circled cells responded by a cytoplasmic pH-drop, whereas with ‘reverse polarity’ (stimulus at t = 120 s) the top electrode is the cathode and the red circled cells responded. Bottom - additional illumination: Light spectrum details are provided in Methods section. Regions sampled are circled in the MiCAM image. Images were acquired at 100 ms intervals over a time period of 546 s. Duration of light pulses (on/off) was 10 s. Abbreviations: fr, far red; nr, near red; c, cyan; b, blue; v, violet. Under blue and violet illumination, ArcLight decreases in fluorescence intensity due to photobleaching, which is more pronounced when light of high energy (violet = 390 nm) is used as compared to lower energy (blue = 438 nm). The recovery of fluorescence after the bleaching light has been switched off is due to diffusion of unbleached fluorescent proteins into the focal plane of the imaging objective, an effect known as FRAP (Fluorescence recovery after photo bleaching). The small increases in the signal during illumination with far red, near red and cyan result from insufficient spectral separation of the illuminating light from the optical emission path of the microscope