Figure 5

High-resolution protein location determination upon organelle swelling. (a) Single slice images of a COS-7 cell co-expressing TOMM20-GFP (green) and PARL-mCherry (red) pre (culture medium, top row) and post incubation in diH₂O for 200 s (diH₂O, bottom row). Scale bar, 0.5 µm. (a,b,e,f) The asterisks (*) (column 1) indicate the positions of mitochondrial matrix surrounded by swollen mitochondria membrane (a, b and f, bottom row) or ER lumen surrounded by ER membrane (e and f, rows 1 and 2). (a,c,d,e) Profiles of fluorescence intensity (arbitrary units) taken along the white dotted lines (a,c,d, column 3, e, column 1) are presented (right). (b) Single slice images of a COS-7 cell (top row) and a U2-OS cell (bottom row) co-expressing TOMM20-GFP (green) and PARL-mCherry (red) post incubation in diH₂O for 200 s. The additional PARL-mCherry IMM structures are indicated (yellow arrowheads). Scale bars, 0.5 µm. (c) Single slice images of a HeLa cell co-expressing PARL-GFP (green) and PINK1-mCherry (red) pre (culture medium, top row) and post incubation in diH₂O for 200 s (diH₂O, bottom row). Scale bars, 1 µm. (d) MIP (columns 1 and 2) and single slice images (column 3) of a HEK cell co-expressing wtPrP-GFP (green) and GalT-RFP (red). (Columns 2 and 3) Images are 2.7x magnifications from the corresponding area outlined in column 1 (yellow rectangle). Golgi signals are shown pre (culture medium, top row) and post incubation in diH₂O for 240 s (diH₂O, bottom row). Scale bars, 2 µm. (e) Single slice images of a HeLa cell expressing (top) mCherry-CD3δ-linker-YFP or (bottom) co-expressing wtPrP-GFP (green) and CD3δ-mCherry (red) post incubation in diH₂O for 200 s. (Top) The mCherry signal of mCherry-CD3δ-linker-YFP is shown in red and the YFP signal in green. (Bottom) For better orientation, an area of swollen ER next to the plasma membrane (yellow arrowhead) is displayed. Scale bars, 0.5 µm. (f) STED (green) and confocal (red) single slice images of HeLa cells co-expressing TAP1-GFP (rows 1 and 2) and TOMM20-GFP (row 3) post incubation in diH₂O for 200 s. Shown are images of cells fixed in methanol (MeOH) (top row) or paraformaldehyde (PFA) (rows 2 and 3). Scale bars, 0.5 µm.