Figure 2.

Spatial analysis of exocytic hot spots. A, Fluorescence responses (averaged over the entire terminal) to four stimulus trains (each 100 Hz for 0.5 s). Inset, Image of resting terminal. The bright spot near the center (*) showed no fluorescence increase during nerve stimulation. B, Fluorescence increases (difference between average of last 3 control images before train and average of 3 images at the peak of fluorescence) are shown for each of the four trains on a pixel-by-pixel basis. Regions of large increases (red) tended to recur at the same positions for each train. Scale bar, 10 μm. C, The images from B were aligned, and pixels at the same positions within the boundaries of the terminal (n = 3806 pixels) were averaged. The 50 positions with the highest average fluorescence increase were identified, and the individual pixel values from each train were plotted (red). The same procedure was followed for the positions with lowest (blue) and middle (green) rises in fluorescence. The graph shows that pixel values in one train predict pixel values in all trains. D, The mean-variance scatter plot for each pixel position shows a reasonably strong correlation (r² = 0.59), again suggesting consistent responses from train to train. E, The fluorescence of three spots are plotted. The spot with the highest fluorescence at the outset (* in A) showed no response at all during stimulation, whereas the much dimmer spots showed robust responses. F, Pixel-by-pixel scatter plot of initial background fluorescence versus rise in fluorescence during stimulation shows virtually no correlation (r² = 0.02). a.u., Arbitrary units.