(A) In vitro prepared COPI vesicles labeled with Alexa488 were attached to glass coverslips. Immunofluorescence against COPI was performed, and samples were imaged by both confocal and STED microscopy. The size of COPI vesicles was determined by STED microscopy using a custom Matlab routine by fitting to a 2D Lorenztian function and confocal images were fit to a 2D Gaussian function. Graph, size distribution of 64 in vitro COPI vesicles fit with a 2D Lorentzian function. The mean is 114 nm. (B) HeLa cells expressing ssGFP-FM4-CD8, ST-RFP and VSV-G were incubated at 20°C in the presence of AP21998 and CHX for 2 hr, fused, and fixed after 30 min. Immunofluorescence was performed against β-COP to label all the Golgi related structures. COPI like transport intermediates imaged by STED microscopy were identified based on size and the contents were evaluated. Numbered boxes illustrating types of cargo containing COPI like intermediates. Red squares, different objects at low and high magnification. The sizes of the intermediates were determined using a 2D Lorentzian function. Graph (C) shows the size distribution of 179 carriers fit with a 2D Lorentzian function, The mean is 128 nm. Graph (D) shows the distribution of COPI spots positive for either ssGFP-FM4-CD8 cargo or ST-RFP, or both (gray columns). As a positive control we used cells co-expressing ssRFP-FM4-CD8 instead of ST-RFP (white columns). Control: 48 cells, 138 spots analyzed; Experimental: 31 cells, 179 spots analyzed. Results are representative of two independent experiments, each one being duplicated.