Figure 2: Patterns of two Sb2-associated fluorophores reveal nonuniform vesicular distribution of Sb2.

(a–d) Each panel contains (left to right) a SIM image, a graph and a drawing. SIM images (left column) show different patterns of Atto (red) and YpH (green) fluorophores tagging Sb2. Graphs (middle column) show normalized/relative fluorescence intensity profiles (red and green fluorescence along the section, that is, line, in corresponding SIM image). The right ordinates of the intensity profiles show absolute intensity (a.u., arbitrary unit) of green and red fluorescent puncta. Schematics in the right column show possible arrangements of two fluorophores in a single vesicle. The arrow and vertical dash-line are drawn to visualize the top view of the fluorophore pair (c). The order of images (a–d) is according to abundance (shown in %) of the pattern observed in astrocytes as summarized in graph (e). The error bars represent s.e.m. We analyzed three astrocytes in which a total of 349 vesicles were examined. *P<0.02 and **P<0.001 (ANOVA). As indicated in line profile in a, distance (proximity) between the green and red fluorophores, d, can be determined as the distance between the peaks. The frequency distribution plot of inter-fluorophore distance measured for all YpH-Atto pairs is shown in f. The mean value for the distance between YpH and Atto is 65±2 nm (mean±s.e.m.), which is calculated by fitting a Gaussian curve (f) on a frequency distribution plot of the form Counts/bin={A/[σ(✓2π)]} × exp{−[x−μ)²/2σ²]} (Equation 1) where x=distance (nm), total count A=(4,295±503); σ=28.6; μ=(65±2) nm. The squared correlation coefficient R²=0.90, n=541 vesicles, 5 cells; difference from zero was statistically significant (P<0.001; ANOVA).