Figure 5
Schematic of overall image processing strategy using Fiji/ImageJ and example images of auto alignment process using MetaXpress and Fiji/ImageJ. (a) Raw images from different time points are first aligned, then overlapping regions of interest (ROIs) from multiple images are selected for further analysis. In aligned ROIs the number of presynaptic VAMP2 and postsynaptic PSD95 puncta are counted separately. Then processed ROIs of pre- and postsynaptic puncta from the same field are merged and the numbers of colocalized puncta are counted. See Fig. 3 for example images; (b) Example of image alignment: raw images from approximately the same position on the plate are collected serially over time (Top row: VAMP2 presynaptic puncta from DIV 21 to 30). The images are aligned (middle) and aligned regions of interest are prepared for analysis (Bottom row: same VAMP2 presynaptic puncta from DIV 21 to 30 after alignment).
