Figure 3.
Figure 3. Live CLEM analysis of maturing Golgi cisternae harboring different markers. (A) 3D images of WT cells expressing Mnn9-mCherry (cis-Golgi, red) and Sys1-2xGFP (trans-Golgi, green) were reconstructed from 61 optical slices 100 nm apart taken at 9.85-s intervals. A white dotted circle indicates a cisterna maturing from cis to trans, in which cis- and trans-Golgi markers appeared to localize in a spatially segregated fashion. Glutaraldehyde was added at 30.0 s, and the image of the fixed cell was obtained at 66.1 s. Scale bar, 1 µm. (B) CLEM images of the cell indicated in A. Left panel shows a section image of fluorescence signals superimposed with the TEM image of the corresponding section. Black arrowheads indicate cis-Golgi cisternae. Black dotted circles show the same cisterna as indicated by the white dotted circle in A. Scale bar, 500 nm. Right panels show serial sections of CLEM (upper) and TEM (lower) images of selected cisternae (boxed area in the left panel). Scale bar, 200 nm. (C) Enlarged 3D reconstructed images of the maturing cisterna and a cis-Golgi cisterna by SCLIM (upper) and TEM (lower). The yellow structure in TEM 3D indicates the maturing Golgi cisterna, and the red indicates a neighboring cis-Golgi cisterna. Continuous membrane structure was observed in all of three independent maturing cisternae. Scale bar, 200 nm. Full 3D reconstruction of the SCLIM and TEM images is animated in Video 2. (D) 3D images of WT cells expressing mRFP-Sed5 (cis-Golgi, red) and Sec7-GFP (TGN, green) were reconstructed from 81 optical slices 80 nm apart of cell taken at 10.8-s intervals. White dotted circles indicate a maturing cisterna from cis to TGN, whose markers appeared to localize in a segregated fashion. Glutaraldehyde was added after 21.6 s, and the cell was judged to be fixed between 32.6 and 43.6 s. Scale bar, 1 µm. (E) CLEM images of the cell indicated in D. Left panel shows the section image of fluorescence signals superimposed with the TEM image of the corresponding section. Black and white arrowheads indicate cis-Golgi and TGN cisternae, respectively. Black dotted circles show the same cisterna indicated by the white dotted circle in D. Scale bar, 500 nm. Right panels show serial sections of CLEM (upper) and TEM (lower) images of the selected cisterna (boxed area in the left panel). Scale bar, 200 nm. (F) 3D reconstructed images of the maturing cisterna by SCLIM (upper) and TEM (upper second). Lower two images indicate the same 3D reconstructed images shown at another angle. Scale bar, 200 nm. Full 3D reconstruction of SCLIM and TEM images is animated in Video 3.

Live CLEM analysis of maturing Golgi cisternae harboring different markers. (A) 3D images of WT cells expressing Mnn9-mCherry (cis-Golgi, red) and Sys1-2xGFP (trans-Golgi, green) were reconstructed from 61 optical slices 100 nm apart taken at 9.85-s intervals. A white dotted circle indicates a cisterna maturing from cis to trans, in which cis- and trans-Golgi markers appeared to localize in a spatially segregated fashion. Glutaraldehyde was added at 30.0 s, and the image of the fixed cell was obtained at 66.1 s. Scale bar, 1 µm. (B) CLEM images of the cell indicated in A. Left panel shows a section image of fluorescence signals superimposed with the TEM image of the corresponding section. Black arrowheads indicate cis-Golgi cisternae. Black dotted circles show the same cisterna as indicated by the white dotted circle in A. Scale bar, 500 nm. Right panels show serial sections of CLEM (upper) and TEM (lower) images of selected cisternae (boxed area in the left panel). Scale bar, 200 nm. (C) Enlarged 3D reconstructed images of the maturing cisterna and a cis-Golgi cisterna by SCLIM (upper) and TEM (lower). The yellow structure in TEM 3D indicates the maturing Golgi cisterna, and the red indicates a neighboring cis-Golgi cisterna. Continuous membrane structure was observed in all of three independent maturing cisternae. Scale bar, 200 nm. Full 3D reconstruction of the SCLIM and TEM images is animated in Video 2. (D) 3D images of WT cells expressing mRFP-Sed5 (cis-Golgi, red) and Sec7-GFP (TGN, green) were reconstructed from 81 optical slices 80 nm apart of cell taken at 10.8-s intervals. White dotted circles indicate a maturing cisterna from cis to TGN, whose markers appeared to localize in a segregated fashion. Glutaraldehyde was added after 21.6 s, and the cell was judged to be fixed between 32.6 and 43.6 s. Scale bar, 1 µm. (E) CLEM images of the cell indicated in D. Left panel shows the section image of fluorescence signals superimposed with the TEM image of the corresponding section. Black and white arrowheads indicate cis-Golgi and TGN cisternae, respectively. Black dotted circles show the same cisterna indicated by the white dotted circle in D. Scale bar, 500 nm. Right panels show serial sections of CLEM (upper) and TEM (lower) images of the selected cisterna (boxed area in the left panel). Scale bar, 200 nm. (F) 3D reconstructed images of the maturing cisterna by SCLIM (upper) and TEM (upper second). Lower two images indicate the same 3D reconstructed images shown at another angle. Scale bar, 200 nm. Full 3D reconstruction of SCLIM and TEM images is animated in Video 3.

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