land-ExM coupled with immunostaining LR-ExM for lipid vesicle identification. (A–C) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-Lamp2 antibodies (yellow). The anti-Lamp2 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 lysosomes. Scale bar: 500 nm in pre-expansion unit. (H) Intensity profile along the gray line across the lysosome in image (D). (I–K) land-ExM lipid (magenta) and protein (green) images of U2OS cells immunostained with anti-clathrin antibodies (yellow). The anti-clathrin 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 images of I–K showing details of clathrin-coated pits. Scale bar: 500 nm in pre-expansion unit. (P) Intensity profile along the gray line across the clathrin-coated pit in image (L). All images were taken with an Airyscan microscope.