Figure 4

Automated image processing and synapse quantification using MetaXpress and Fiji/ImageJ. Example of image processing steps from raw image to processed, ready-to-analyze image for VAMP2-mRFP (a) and PSD95-mVenus (b). Raw images were processed using ‘Flatten Background’, 2D Deconvolution using ‘No Neighbors’, morphology filtering using ‘Top-Hat’, and binarized; (c) Example of transmitted light image and processed transmitted light image; (d) Merged images of transmitted light, VAMP-mRFP, and PSD95-mVenus, after processing, all synapses that will be used for quantification are localized on the neurite. Note that in the selected region, almost all PSD95-mVenus are colocalized with VAMP-mRFP on one neurite, while on the other neurite only VAMP-mRFP are present at the time image was taken.