Three-color 4D observation of Golgi cisternal maturation in living yeast cells. WT cells expressing Mnn9-mCherry (cis-Golgi, red), Sys1-2xGFP (trans-Golgi, green), and Sec7-iRFP (TGN, blue) were observed by SCLIM. (A) Representative 3D images of merged (cis, trans, and TGN markers, upper) and each marker (lower) are shown. Scale bar, 1 µm. (B) 3D merged images of cis and trans, cis and TGN, and trans and TGN markers are shown (upper). Scale bar, 1 µm. Lower panels show enlarged images of selected cisternae (boxed areas in upper panels). Scale bar, 250 nm. (C) A representative 3D image of cisternae is shown on the left. Scale bar, 1 µm. Right panels show 4D (3D time-lapse) images of a selected cisterna (boxed area in the left panel). 3D images were reconstructed from 31 optical slices 100 nm apart around the center of the cell taken at 6.25-s intervals. Merged (cis, trans, and TGN markers) and each marker’s images are shown. Scale bar, 500 nm. (D) Relative fluorescence intensity changes of cis, trans, and TGN markers in the selected cisterna in C are shown. Statistical data are shown in Fig. 2 C.