Fig. 1: On-section copper-catalysed click reaction for visualizing lipids using CLEM.

a, The chemical structure of the bifunctional lipid probe PC(16:0|Y). b, The Lipid-CLEM workflow: cells are loaded with bifunctional lipid probes, probes are UV crosslinked and samples are high-pressure frozen. Lipids are fluorescently labelled directly on sections after resin embedding via freeze substitution. Samples are imaged by FM and EM. The images are overlaid by correlation. c, Cells were co-labelled with fluorescent LDL (blue) and Tf (magenta). The lipid signal is shown in orange. Control stain: cells with (+lipid +UV) and without (−lipid −UV) bifunctional PC(16:0|Y) were chemically fixed, permeabilized, labelled by click chemistry and imaged using confocal FM. On-section stain: thin sections (100 nm) of HM20 samples treated with (+lipid +UV) and without (−lipid −UV) bifunctional PC(16:0|Y) were labelled according to the workflow. The dataset includes three independent biological replicates that yielded similar results. Multicoloured TetraSpecs are indicated by arrows. The dotted red lines represent cell borders if not visible otherwise. Scale bars, 10 µm (overview images) and 1 µm (ROIs). d, CLEM of thin HM20 sections (100 nm) shows localization of the lipid at the plasma membrane and endosomes. The fluorescent overview image (FM), medium magnified TEM image (EM), overlay and correlation error map of 100-nm-thick sections are shown left to right. Representative ROIs for an endosome (1) and the plasma membrane (2) are shown, acquired at higher magnification by TEM. Scale bar, 10 µm (overview images) and 1 µm (ROIs). The HM20 dataset includes one replicate.