Figure 6:

EOF onset and offset latency quantification using fluorescence measurement. (a) Frame showing the tip of the micropipette when no voltage was applied. A region of interest (ROI) was fixed near the micropipette outlet (red circle) with an area of 80 μm² for measuring mean grayscale value (GV) in the region. (b-d) Maximum fluorescein bolus formation when 2, 5 and 10 V pulse was applied. (e) Change in GV within the ROI for a single representative pulse of 2, 5 and 10 V. (f) To capture the GV trend (green data points) the curve was smoothed using a Savitzky-Golay filter and latencies were expressed in terms of full width half maximum (FWHM) estimation. Y-intercepts (time to reach T1 and T2 when V is turned ON at t = 0s) on the filtered curve (red solid line) at half maximum are represented by square data points and peak is represented by a solid upright triangle (time duration Tp). (g) Comparison of boxplots containing lower quartile (bottom of box), upper quartile (top of box) and median (black line) of T1 latencies obtained from 2, 5, and 10 V injection pulses (n = 57 for each voltage value). A significant (p<0.001 denoted by ‘***’ and p<0.05 denoted by ‘*’) decrease in latency between voltage ON and T1 can be observed when voltage is increased from 2 V to 10 V. This indicates that lower glutamate injection onset (or lower T1 latency) can be achieved by using higher voltages.