Fig. 3. Spatiotemporal localisation of Ca²⁺ signals in A. thaliana G-CaMP3 roots upon salinity accumulation. Flow rate was set to 20 μL min⁻¹. Original video files are 1 fps. Control media (1/2 MS/0.31 mM MES) was injected into the bi-dfRC microchannel through inlets A & B (at the shoot site) to create a steady flow for ∼19 minutes, then switched to the desired experimental treatment using a gradient. The gradient was tracked by fluorescence using 0.01 mM HPTS dye (green). All root schematics depict treatment application with the rectangular regions of interest highlighted (refer to key). Bright field (BF) and control (wild type Col-0) roots are displayed on the left of the figure. Scale; F = fluorescence intensity. (a) Following treatment switch to control media, no Ca²⁺ burst indicated by the G-CaMP3 was observed (n = 3). (b) Following switch to 100 mM NaCl, a strong Ca²⁺ signal was detected (n = 3). (c) Line graph with a two-way ANOVA multiple comparisons Tukey's honestly significant difference (HSD) mean comparison test (P-value ≤ 0.05) depicting average fluorescence intensity (grey scale; pixel brightness) of G-CaMP3 within three rectangular regions of interest (tip, maturation/elongation (ME) zone and elongation/differentiation (ED) zone) upon targeted exposure to control treatment (n = 3). (d) Line graph depicting average fluorescence intensity of G-CaMP3 across three rectangular regions upon NaCl treatment (n = 3). Asterisks (*) indicate statistical significance.