Figure 1: Vesicle-associated membrane protein Sb2 fluorescently labelled at the luminal and cytoplasmic sides of the vesicle membrane.

(a) The diagram represents an astrocytic vesicle (left) in which a version of Sb2 is expressed, with a fluorescent protein in the vesicle lumen (fluorophore A, that is, YpH) and with an immunolabelled cytoplasmic domain (fluorophore B, that is, Atto 594). The boxed diagram on right shows the architecture of Sb2 across the vesicle membrane, drawn to scale by considering the length of Sb2^18,19. b and c are images from CLSM and SIM, respectively. To stain Sb2 in vesicle lumen, astrocytes were transfected with a plasmid encoding YSpH (green). To stain the N-terminal part of Sb2 located on the cytosolic side of the vesicle membrane, primary antibody against Sb2 and a secondary antibody conjugated to Atto 594 (Atto; red) were used. Merge panels show overlay of YSpH and Atto signals arising from the vesicle lumen and the cytoplasm. Mask panels show only the colocalized pixels between YpH and Atto. Bar, 10 μm. Note the smaller amount of colocalization (percent, shown for representative single astrocytes) in the SIM image than in the CLSM image.