Cargo transfer from the early to the late zone is prevented in the cells deficient in COPI function. ret1-1 cells expressing heat-shock–inducible Axl2-GFP (cargo, green) and constitutive mRFP-Sed5 (cis-Golgi, red) and Sec7-iRFP (TGN, blue) were incubated at 37°C for 30 min and then observed on the stage at 37°C by SCLIM. (A) A representative 3D image is shown on the left. Axl2-GFP was detected on the Golgi cisternae labeled with mRFP-Sed5 and Sec7-iRFP. Scale bar, 1 µm. Right panels show the time-lapse 3D images of the selected cisternae shown in boxes in A. 3D images were reconstructed from 16 optical slices 200 nm apart taken at 5.4-s intervals. Merged (cargo, cis, and TGN) and individual images are shown. Scale bar, 250 nm. (B) Quantification of Golgi/TGN markers and cargo Axl2 during the observation period. Fluorescence intensities of cargo, cis-Golgi, and TGN markers of five selected cisternae were quantified with Z-stacks collected every 9.2 s. The fluorescence intensities of mRFP-Sed5, Sec7-iRFP, or Axl2-GFP at time 0 in each of the five experiments were set to 1. Mean values of all the experiments per each time point were subjected to statistical analysis. Bars represent standard errors of means. (C) The panels in the first and the third rows show enlarged merged images of cargo (green), cis-Golgi (red), and TGN (blue) markers at indicated times of cisternae #1 and #2 of A, respectively. The graphs of the second and fourth rows show relative fluorescence profiles of cargo (green), cis-Golgi (red), and TGN (blue) markers along the white lines in the cisterna shown above. Note that most of cargo stayed in the cis-Golgi zone (arrowheads) and a small portion of cargo appeared in the TGN zone occasionally and transiently (arrows). Scale bar, 250 nm.