Figure 4.

Puff sites show little or no movement over periods of several seconds. (a) Panels show average fluorescence images obtained during six successive Ca²⁺ puffs occurring at the same site. Each image is an average of 61–151 frames (420 frames s⁻¹) captured during individual events. Cross-hairs mark the centroid location of the first puff. (b) The top trace plots average fluorescence ratio (ΔF/F₀) measured within a 3 × 3 pixel region (1 × 1 μm) centered on the same puff site as in panel a, showing six discrete events within ∼10 s. The fluorescence images during these puffs were fit to a two-dimensional Gaussian function, and the center positions of the Gaussian fits are plotted as x and y locations at corresponding times (lower traces). Solid circles show locations derived from individual frames, and triangles mark the mean locations (mean ± SE) during each of the six puffs. (c) Plot of the mean-square displacement from origin versus time for all puff centroids analyzed (n = 14 puff sites). The line is a linear regression fit, with a slope corresponding to a two-dimensional diffusion coefficient of 0.002 μm² s⁻¹. (d) Diffusion coefficients calculated for 14 separate puff sites (solid circles), and overall mean ±1 SE (open square).