Dynamics of three Golgi markers Mnn9-mCherry (cis-Golgi), Sys1-2xGFP (trans-Golgi), and Sec7-iRFP (TGN). WT cells expressing Mnn9-mCherry (cis-Golgi, red), Sys1-2xGFP (trans-Golgi, green), and Sec7-iRFP (TGN, blue) were observed by SCLIM. (A and B) Representative 3D images of cisternae are shown on the top. Scale bar, 1 µm. Middle panels show 4D (3D time-lapse) images of selected cisternae (boxed area in the top panels). 3D images were reconstructed from 31 optical slices 200 nm apart around the center of the cell taken at 4.7-s intervals. The frames where Mnn9-mCherry disappeared were set as time 0. Merged (cis, trans, and TGN markers) and each marker’s images are shown. The cis-Golgi signals seen at ∼75.2 s in B were due to the fluorescence of another cis-Golgi cisterna behind the selected cisterna as shown by the 3D-rotated image (rightmost). Scale bar, 500 nm. Lower panels show relative fluorescence intensity changes of cis, trans, and TGN markers in the selected cisternae in middle panels. (C) Compiled relative fluorescence intensities of three markers (top), Mnn9-mCherry (second), Sys1-2xGFP (third), and Sec7-iRFP (bottom) over time from seven independent cisternae are shown. The frames where Mnn9-mCherry signals disappeared were set as time 0.