land-ExM coupled with immunostaining LR-ExM for membrane-bound organelle visualization. (A–C) land-ExM total lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Tom20 antibodies (yellow). The anti-Tom20 antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (D–G) Magnified images of A–C showing details of mitochondria. Scale bar: 1 µm in pre-expansion unit. (H) Intensity profile along the cyan line across the mitochondria in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Sec61b antibodies (yellow). The anti-Sec61b antibodies are labeled LR-ExM second antibodies, which are second antibodies conjugated with NHS-digoxigenin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4. (L–O) Magnified in images of I–K showing details of ER. Scale bar: 1 µm in pre-expansion unit. (P) Intensity profile along the cyan line across the ER in image (L). All images were taken with an Airyscan microscope.